Methods of diagnosing cervical cancer

ABSTRACT

The invention provides reagents and methods for detecting pathogen infections in human samples. This detection utilizes specific proteins to detect the presence of pathogen proteins or abnormal expression of human proteins resulting from pathogen infections. Specific methods, compositions and kits are disclosed herein for the detection of oncogenic Human papillomavirus E6 proteins in clinical samples.

CROSS-REFERENCE

This application is a divisional of U.S. patent application Ser. No.10/630,590, filed Jul. 29, 2003, which application: a) claims thebenefit of U.S. Provisional Application No. 60/409,298, filed Sep. 9,2002, and U.S. Provisional Application No. 60/450,464, filed Feb. 27,2003 b) is a CIP of PCT Application No. US02/24655, filed Aug. 2, 2002,which application claims the benefit of U.S. Provisional Application No.60/309,841 filed Aug. 3, 2001, and U.S. Provisional Application No.60/360,061, filed Feb. 25, 2002 c) is a CIP of U.S. Non-Provisionalapplication Ser. No. 10/080,273, filed Feb. 19, 2002, which applicationclaims the benefit of U.S. Provisional Application No. 60/269,523, filedFeb. 16, 2001, and d) is a CIP of U.S. Non-Provisional application Ser.No. 09/710,059, filed Nov. 10, 2000, all of which applications areincorporated herein by reference in their entirety.

ACKNOWLEDGMENT OF GOVERNMENT SUPPORT

This invention was made with Government support under Small BusinessInnovation Research Grant No. IR43CA103383-01, awarded by the NationalCancer Institute. The Government may have certain rights in thisinvention.

FIELD OF THE INVENTION

The present invention relates to detection of biological markers frompathogenic organisms, such as observed in certain human Papillomavirus(HPV) infections, and methods for using such diagnostics to identifysamples that are infected and may lead to cancerous growth or otherdisorders. The present invention also discloses composition, methods andkits for the detection of oncogenic HPV E6 proteins in clinical samplesas a cancer diagnostic.

BACKGROUND OF THE INVENTION

Cervical cancer is the second most common cancer diagnosis in women andis linked to high-risk human papillomavirus infection 99.7% of the time.Currently, 12,000 new cases of invasive cervical cancer are diagnosed inUS women annually, resulting in 5,000 deaths each year. Furthermore,there are approximately 400,000 cases of cervical cancer and close to200,000 deaths annually worldwide. Human papillomaviruses (HPVs) are oneof the most common causes of sexually transmitted disease in the world.Overall, 50-75% of sexually active men and women acquire genital HPVinfections at some point in their lives. An estimated 5.5 million peoplebecome infected with HPV each year in the US alone, and at least 20million are currently infected. The more than 100 different isolates ofHPV have been broadly subdivided into high-risk and low-risk subtypesbased on their association with cervical carcinomas or with benigncervical lesions or dysplasias.

A number of lines of evidence point to HPV infections as the etiologicalagents of cervical cancers. Multiple studies in the 1980's reported thepresence of HPV variants in cervical dysplasias, cancer, and in celllines derived from cervical cancer. Further research demonstrated thatthe E6-E7 region of the genome from oncogenic HPV 18 is selectivelyretained in cervical cancer cells, suggesting that HPV infection couldbe causative and that continued expression of the E6-E7 region isrequired for maintenance of the immortalized or cancerous state. Thefollowing year, Sedman et al demonstrated that the E6-E7 genes from HPV16 were sufficient to immortalize human keratinocytes in culture.Barbosa et al demonstrated that although E6-E7 genes from high risk HPVscould transform cell lines, the E6-E7 regions from low risk, ornon-oncogenic variants such as HPV 6 and HPV 11 were unable to transformhuman keratinocytes. More recently, Pillai et al examined HPV 16 and 18infection by in situ hybridization and E6 protein expression byimmunocytochemistry in 623 cervical tissue samples at various stages oftumor progression and found a significant correlation betweenhistological abnormality and HPV infection.

Current treatment paradigms are focused on the actual cervical dysplasiarather than the underlying infection with HPV. Women are screened byphysicians annually for cervical dysplasia and are treated withsuperficial ablative techniques, including cryosurgery, laser ablationand excision. As the disease progresses, treatment options become moreaggressive, including partial or radical hysterectomy, radiation orchemotherapy. A significant unmet need exists for early and accuratediagnosis of oncogenic HPV infection as well as for treatments directedat the causative HPV infection, preventing the development of cervicalcancer by intervening earlier in disease progression. Humanpapillomaviruses characterized to date are associated with lesionsconfined to the epithelial layers of skin, or oral, pharyngeal,respiratory, and, most importantly, anogenital mucosae. Specific humanpapillomavirus types, including HPV 6 and 11, frequently cause benignmucosal lesions, whereas other types such as HPV 16, 18, and a host ofother strains, are predominantly found in high-grade lesions and cancer.Individual types of human papillomaviruses (HPV) which infect mucosalsurfaces have been implicated as the causative agents for carcinomas ofthe cervix, anus, penis, larynx and the buccal cavity, occasionalperiungal carcinomas, as well as benign anogenital warts. Theidentification of particular HPV types is used for identifying patientswith premalignant lesions who are at risk of progression to malignancy.Although visible anogenital lesions are present in some persons infectedwith human papillomavirus, the majority of individuals with HPV genitaltract infection do not have clinically apparent disease, but analysis ofcytomorphological traits present in cervical smears can be used todetect HPV infection. Papanicolaou tests are a valuable screening tool,but they miss a large proportion of HPV-infected persons due to theunfortunate false positive and false negative test results. In addition,they are not amenable to worldwide testing because interpretation ofresults requires trained pathologists.

Conventional viral detection assays, including serologic assays,sandwich ELISA assays and growth in cell culture, are not commerciallyavailable and/or are not suitable for the diagnosis and tracking of HPVinfection. Recently, several PCR (polymerase chain reaction)-based testsfor HPV infections have become available. Though the tests provide thebenefit of differentiating oncogenic from non-oncogenic infections, theyare fairly expensive to administer and require highly trainedtechnicians to perform PCR and/or luminometer assays. In addition, PCRhas a natural false positive rate that may invoke further testing orprocedures that are not required. Since the oncogenicity of HPV has beenshown to be protein based, early detection of HPV DNA or RNA may lead tounnecessary medical procedures that the body's immune system may solvenaturally.

The detection and diagnosis of disease is a prerequisite for thetreatment of disease. Numerous markers and characteristics of diseaseshave been identified and many are used for the diagnosis of disease.Many diseases are preceded by, and are characterized by, changes in thestate of the affected cells. Changes can include the expression ofpathogen genes or proteins in infected cells, changes in the expressionpatterns of genes or proteins in affected cells, and changes in cellmorphology. The detection, diagnosis, and monitoring of diseases can beaided by the accurate assessment of these changes. Inexpensive, rapid,early and accurate detection of pathogens can allow treatment andprevention of diseases that range in effect from discomfort to death.

The following publications are of interest: Munger (2002) Front. Biosci.7:d641-9; Glaunsinger (2000) Oncogene 19:5270-80; Gardiol (1999)Oncogene 18:5487-96; Pim (1999) Oncogene 18:7403-8; Meschede (1998) J.Clin. Microbiol. 36:475-80; Kiyono (1997.) Proc. Natl. Acad. Sci.94:11612-6; and Lee (1997) Proc. Natl. Acad. Sci. 94:6670-5. Inaddition, the following patents and patent applications are of interest:Bleul, U.S. Pat. No. 6,322,794; Cole, U.S. Pat. No. 6,344,314;Schoolnik, U.S. Pat. No. 5,415,995; Bleul, U.S. Pat. No. 5,753,233;Cole, U.S. Pat. No. 5,876,723; Cole, U.S. Pat. No. 5,648,459; Orth, U.S.Pat. No. 6,391,539; Orth, U.S. Pat. No. 5,665,535; Schoolnik, U.S. Pat.No. 4,777,239.

SUMMARY

Methods and compositions for detection of proteins from pathogens thatmay result in oncogenic cellular transformation or biologicalabnormalities in a variety of cell types (e.g., cervical, anal, penile,throat) are provided herein. These methods and compositions can beutilized to detect the presence of pathogens including, but not limitedto, those that result in diseases such as cervical cancer, penilecancer, anal cancer and throat cancer, for example. More specifically,methods, compositions and kits are described for the detection ofoncogenic HPV E6 proteins in clinical samples

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a bar graph showing that PDZ proteins can specificallyrecognize oncogenic E6 proteins from human papillomavirus. An ELISAassay was used to demonstrate that a PDZ protein (TIP-1) couldspecifically recognize full length E6 protein from an oncogenic strain(HPV18) but did not show any reactivity with a non-oncogenic strain(HPV11). Series 1 and Series 2 represent independent trials. E6 abindicates that an antibody against E6 from HPV18 was used for detectioninstead of the PDZ protein.

FIG. 2 is a line graph showing that PDZ binding to HPV 18 E6 PLs istemperature dependent. This Figure uses a modified ELISA to determinebinding of the PDZ domains of TIP-1 or MAGI-1 (domain 2) to a peptidecorresponding to the C-terminal 20 AA of the E6 protein from HPV18.Numbers in the legend represent independent experiments. −RT indicatesthat the association was carried out at room temperature. Data serieslacking −RT were allowed to associate at 4° C.

FIG. 3 is a line graph showing anti-HPV18E6 antibody recognition ofGST-HPV18E6 fusion protein. Day 28 sera from a Balb/c mouse immunizedwith HPV18E6 protein was tested for reactivity to either GST-HPV18E6protein or GST alone.

FIG. 4 (A-D) is a panel of four line graphs showing the effect of lysateupon ability of recombinant E6 protein from HPV type 16 to binddifferent PDZ domains.

FIG. 5 (A-B) is an autoradiograph showing that several PDZ domains canbind and coprecipitate oncogenic E6 proteins from cells.

FIG. 6 is an autoradiograph showing the results of a western blotdemonstrating detection of endogenous HPV16 E6 protein in the SiHacervical cancer line.

FIG. 7 is an autoradiograph showing that HPV 16 E6 protein can bedetected in CasKi and SiHa cervical cancer cell lines by western blots,and detection is enhanced when lysates are made in the presence ofProteasome inhibitor.

FIG. 8 is a line graph showing ELISA detection of HPV16 E6 protein inSiHa and CasKi cervical cell lines.

FIG. 9: is an autoradiograph showing dot blot detection of HPV16 E6protein in cell lysates.

FIG. 10: is an autoradiograph showing dot blot detection of endogenousHPV16 E6 protein in lysates of SiHa and CasKi cervical cell lines.

DETAILED DESCRIPTION I. Definitions

A “marker” or “biological marker” as used herein refers to a measurableor detectable entity in a biological sample. Examples or markers includenucleic acids, proteins, or chemicals that are present in biologicalsamples. One example of a marker is the presence of viral or pathogenproteins or nucleic acids in a biological sample from a human source.

As used herein the term “isolated” refers to a polynucleotide, apolypeptide, an antibody, or a host cell that is in an environmentdifferent from that in which the polynucleotide, the polypeptide, theantibody, or the host cell naturally occurs. A polynucleotide, apolypeptide, an antibody, or a host cell which is isolated is generallysubstantially purified. As used herein, the term “substantiallypurified” refers to a compound (e.g., either a polynucleotide or apolypeptide or an antibody) that is removed from its natural environmentand is at least 60% free, preferably 75% free, and most preferably 90%free from other components with which it is naturally associated. Thus,for example, a composition containing A is “substantially free of” Bwhen at least 85% by weight of the total A+B in the composition is A.Preferably, A comprises at least about 90% by weight of the total of A+Bin the composition, more preferably at least about 95% or even 99% byweight.

The term “biological sample” encompasses a variety of sample typesobtained from an organism and can be used in a diagnostic or monitoringassay. The term encompasses blood and other liquid samples of biologicalorigin, solid tissue samples, such as a biopsy specimen or tissuecultures or cells derived therefrom and the progeny thereof. The termencompasses samples that have been manipulated in any way after theirprocurement, such as by treatment with reagents, solubilization, orenrichment for certain components. The term encompasses a clinicalsample, and also includes cells in cell culture, cell supernatants, celllysates, serum, plasma, biological fluids, and tissue samples. The term“biological sample” is meant to distinguish between a sample in aclinical setting from a sample that may be a recombinant sample orderived from a recombinant sample.

A “fusion protein” or “fusion polypeptide” as used herein refers to acomposite protein, i.e., a single contiguous amino acid sequence, madeup of two (or more) distinct, heterologous polypeptides that are notnormally fused together in a single amino acid sequence. Thus, a fusionprotein can include a single amino acid sequence that contains twoentirely distinct amino acid sequences or two similar or identicalpolypeptide sequences, provided that these sequences are not normallyfound together in the same configuration in a single amino acid sequencefound in nature. Fusion proteins can generally be prepared using eitherrecombinant nucleic acid methods, i.e., as a result of transcription andtranslation of a recombinant gene fusion product, which fusion comprisesa segment encoding a polypeptide of the invention and a segment encodinga heterologous protein, or by chemical synthesis methods well known inthe art.

A “fusion protein construct” as used herein is a polynucleotide encodinga fusion protein.

An “oncogenic HPV strain” is an HPV strain that is known to causecervical cancer as determined by the National Cancer Institute (NCI,2001). “Oncogenic E6 proteins” are E6 proteins encoded by the aboveoncogenic HPV strains. Exemplary oncogenic strains are shown in Table 3.

An “oncogenic E6 protein binding partner” can be any molecule thatspecifically binds to an oncogenic E6 protein. Suitable oncogenic E6protein binding partners include a PDZ domain (as described below), anantibody against an oncogenic E6 protein; other proteins that recognizeoncogenic E6 protein (e.g., p53, E6-AP or E6-BP); DNA (i.e., cruciformDNA); and other partners such as aptamers or single chain antibodiesfrom phage display). In some embodiments, detection of more than 1oncogenic E6 protein (e.g., all oncogenic E6 proteins or E6 proteinsfrom HPV strains 16, 18 and 33) is desirable, and, as such, an oncogenicE6 protein binding partner may be antibody that binds to these proteins,a mixture of antibodies that each bind to a different proteins. As isknown in the art, such binding partners may be labeled to facilitatetheir detection. In general, binding partner bind E6 with an bindingaffinity of 10⁻⁵ M or more, e.g., 10⁻⁶ or more, 10⁻⁷ or more, 10⁻⁸ M ormore (e.g., 10⁻⁹ M, 10⁻¹⁰, 10⁻¹¹, etc.).

As used herein, the term “PDZ domain” refers to protein sequence (i.e.,modular protein domain) of less than approximately 90 amino acids,(i.e., about 80-90, about 70-80, about 60-70 or about 50-60 aminoacids), characterized by homology to the brain synaptic protein PSD-95,the Drosophila septate junction protein Discs-Large (DLG), and theepithelial tight junction protein ZO1 (ZO1). PDZ domains are also knownas Discs-Large homology repeats (“DHRs”) and GLGF repeats. PDZ domainsgenerally appear to maintain a core consensus sequence (Doyle, D. A.,1996, Cell 85: 1067-76).

PDZ domains are found in diverse membrane-associated proteins includingmembers of the MAGUK family of guanylate kinase homologs, severalprotein phosphatases and kinases, neuronal nitric oxide synthase, tumorsuppressor proteins, and several dystrophin-associated proteins,collectively known as syntrophins.

Exemplary PDZ domain-containing proteins and PDZ domain sequences areshown in TABLE 2 and EXAMPLE 4. The term “PDZ domain” also encompassesvariants (e.g., naturally occurring variants) of the sequences (e.g.,polymorphic variants, variants with conservative substitutions, and thelike) and domains from alternative species (e.g. mouse, rat). Typically,PDZ domains are substantially identical to those shown in U.S. patentapplication Ser. No. 09/724,553, e.g., at least about 70%, at leastabout 80%, or at least about 90% amino acid residue identity whencompared and aligned for maximum correspondence. It is appreciated inthe art that PDZ domains can be mutated to give amino acid changes thatcan strengthen or weaken binding and to alter specificity, yet theyremain PDZ domains (Schneider et al., 1998, Nat. Biotech. 17:170-5).Unless otherwise indicated, a reference to a particular PDZ domain (e.g.a MAGI-1 domain 2) is intended to encompass the particular PDZ domainand HPV E6-binding variants thereof. In other words, if a reference ismade to a particular PDZ domain, a reference is also made to variants ofthat PDZ domain that bind oncogenic E6 protein of HPV, as describedbelow. In this respect it is noted that the numbering of PDZ domains ina protein may change. For example, the MAGI-1 domain 2, as referencedherein, may be referenced as MAGI-1 domain 1 in other literature. Assuch, when a particular PDZ domain of a protein is referenced in thisapplication, this reference should be understood in view of the sequenceof that domain, as described herein, particularly in the sequencelisting. Table 9, inserted before the claims, shows the relationshipbetween the sequences of the sequence listing and the names and Genbankaccession numbers for various domains, where appropriate.

As used herein, the term “PDZ protein” refers to a naturally occurringprotein containing a PDZ domain. Exemplary PDZ proteins include CASK,MPP1, DLG1, DLG2, PSD95, NeDLG, TIP-33, SYN1a, TIP-43, LDP, LIM, LIMK1,LIMK2, MPP2, NOS1, AF6, PTN-4, prIL16, 41.8 kD, KIAA0559, RGS12,KIAA0316, DVL1, TIP-40, TIAM1, MINT1, MAGI-1, MAGI-2, MAGI-3, KIAA0303,CBP, MINT3, TIP-2, KIAA0561, and TIP-1.

As used herein, the term “PDZ-domain polypeptide” refers to apolypeptide containing a PDZ domain, such as a fusion protein includinga PDZ domain sequence, a naturally occurring PDZ protein, or an isolatedPDZ domain peptide. A PDZ-domain polypeptide may therefore be about 60amino acids or more in length, about 70 amino acids or more in length,about 80 amino acids or more in length, about 90 amino acids or more inlength, about 100 amino acids or more in length, about 200 amino acidsor more in length, about 300 amino acids or more in length, about 500amino acids or more in length, about 800 amino acids or more in length,about 1000 amino acids or more in length, usually up to about 2000 aminoacids or more in length. PDZ domain peptides are usually no more thanabout 100 amino acids (e.g. 50-60 amino acids, 60-70 amino acids, 80-90amino acids, or 90-100 amino acids), and encode a PDZ domain.

As used herein, the term “PL protein” or “PDZ Ligand protein” refers toa naturally occurring protein that forms a molecular complex with aPDZ-domain, or to a protein whose carboxy-terminus, when expressedseparately from the full length protein (e.g., as a peptide fragment of4-25 residues, e.g., 8, 10, 12, 14 or 16 residues), forms such amolecular complex. The molecular complex can be observed in vitro usingthe “A assay” or “G assay” described infra, or in vivo. Exemplary PLproteins listed in TABLES 3 and 4 are demonstrated to bind specific PDZproteins. This definition is not intended to include anti-PDZ antibodiesand the like.

As used herein, a “PL sequence” refers to the amino acid sequence of theC-terminus of a PL protein (e.g., the C-terminal 2, 3, 4, 5, 6, 7, 8, 9,10, 12, 14, 16, 20 or 25 residues) (“C-terminal PL sequence”) or to aninternal sequence known to bind a PDZ domain (“internal PL sequence”).

As used herein, a “PL peptide” is a peptide of having a sequence from,or based on, the sequence of the C-terminus of a PL protein. ExemplaryPL peptides (biotinylated) are listed in TABLE 3.

As used herein, a “PL detector” is a protein that can specificallyrecognize and bind to a PL sequence.

As used herein, a “PL fusion protein” is a fusion protein that has a PLsequence as one domain, typically as the C-terminal domain of the fusionprotein. An exemplary PL fusion protein is a tat-PL sequence fusion.

As used herein, the term “PL inhibitor peptide sequence” refers to PLpeptide amino acid sequence that (in the form of a peptide or PL fusionprotein) inhibits the interaction between a PDZ domain polypeptide and aPL peptide (e.g., in an A assay or a G assay).

As used herein, a “PDZ-domain encoding sequence” means a segment of apolynucleotide encoding a PDZ domain. In various embodiments, thepolynucleotide is DNA, RNA, single stranded or double stranded.

As used herein, the terms “antagonist” and “inhibitor,” when used in thecontext of modulating a binding interaction (such as the binding of aPDZ domain sequence to a PL sequence), are used interchangeably andrefer to an agent that reduces the binding of the, e.g., PL sequence(e.g., PL peptide) and the, e.g., PDZ domain sequence (e.g., PDZprotein, PDZ domain peptide).

As used herein, the terms “agonist” and “enhancer,” when used in thecontext of modulating a binding interaction (such as the binding of aPDZ domain sequence to a PL sequence), are used interchangeably andrefer to an agent that increases the binding of the, e.g., PL sequence(e.g., PL peptide) and the, e.g., PDZ domain sequence (e.g., PDZprotein, PDZ domain peptide).

As used herein, the terms “peptide mimetic,” “peptidomimetic,” and“peptide analog” are used interchangeably and refer to a syntheticchemical compound that has substantially the same structural and/orfunctional characteristics of a PL inhibitory or PL binding peptide ofthe invention. The mimetic can be either entirely composed of synthetic,non-natural analogues of amino acids, or, is a chimeric molecule ofpartly natural peptide amino acids and partly non-natural analogs ofamino acids. The mimetic can also incorporate any amount of naturalamino acid conservative substitutions as long as such substitutions alsodo not substantially alter the mimetic's structure and/or inhibitory orbinding activity. As with polypeptides of the invention which areconservative variants, routine experimentation will determine whether amimetic is within the scope of the invention, i.e., that its structureand/or function is not substantially altered. Thus, a mimeticcomposition is within the scope of the invention if it is capable ofbinding to a PDZ domain and/or inhibiting a PL-PDZ interaction.

Polypeptide mimetic compositions can contain any combination ofnormatural structural components, which are typically from threestructural groups: a) residue linkage groups other than the naturalamide bond (“peptide bond”) linkages; b) non-natural residues in placeof naturally occurring amino acid residues; or c) residues which inducesecondary structural mimicry, i.e., to induce or stabilize a secondarystructure, e.g., a beta turn, gamma turn, beta sheet, alpha helixconformation, and the like.

A polypeptide can be characterized as a mimetic when all or some of itsresidues are joined by chemical means other than natural peptide bonds.Individual peptidomimetic residues can be joined by peptide bonds, otherchemical bonds or coupling means, such as, e.g., glutaraldehyde,N-hydroxysuccinimide esters, bifunctional maleimides,N,N=-dicyclohexylcarbodiimide (DCC) or N,N=-diisopropylcarbodiimide(DIC). Linking groups that can be an alternative to the traditionalamide bond (“peptide bond”) linkages include, e.g., ketomethylene (e.g.,—C(═O)—CH₂— for —C(═O)—NH—), aminomethylene (CH₂—NH), ethylene, olefin(CH═CH), ether (CH₂—O), thioether (CH₂—S), tetrazole (CN₄—), thiazole,retroamide, thioamide, or ester (see, e.g., Spatola (1983) in Chemistryand Biochemistry of Amino Acids, Peptides and Proteins, Vol. 7, pp267-357, A Peptide Backbone Modifications, Marcell Dekker, N.Y.).

A polypeptide can also be characterized as a mimetic by containing allor some non-natural residues in place of naturally occurring amino acidresidues. Nonnatural residues are well described in the scientific andpatent literature; a few exemplary nonnatural compositions useful asmimetics of natural amino acid residues and guidelines are describedbelow.

Mimetics of aromatic amino acids can be generated by replacing by, e.g.,D- or L-naphylalanine; D- or L-phenylglycine; D- or L-2 thieneylalanine;D- or L-1, -2,3-, or 4-pyreneylalanine; D- or L-3 thieneylalanine; D- orL-(2-pyridinyl)-alanine; D- or L-(3-pyridinyl)-alanine; D- orL-(2-pyrazinyl)-alanine; D- or L-(4-isopropyl)-phenylglycine;D-(trifluoromethyl)-phenylglycine; D-(trifluoromethyl)-phenylalanine;D-p-fluorophenylalanine; D- or L-p-biphenylphenylalanine; K- orL-p-methoxybiphenylphenylalanine; D- or L-2-indole(alkyl)alanines; and,D- or L-alkylainines, where alkyl can be substituted or unsubstitutedmethyl, ethyl, propyl, hexyl, butyl, pentyl, isopropyl, iso-butyl,sec-isotyl, iso-pentyl, or a non-acidic amino acids. Aromatic rings of anormatural amino acid include, e.g., thiazolyl, thiophenyl, pyrazolyl,benzimidazolyl, naphthyl, furanyl, pyrrolyl, and pyridyl aromatic rings.

Mimetics of acidic amino acids can be generated by substitution by,e.g., non-carboxylate amino acids while maintaining a negative charge;(phosphono)alanine; sulfated threonine. Carboxyl side groups (e.g.,aspartyl or glutamyl) can also be selectively modified by reaction withcarbodiimides (R═N—C—N—R═) such as, e.g.,1-cyclohexyl-3(2-morpholinyl-(4-ethyl) carbodiimide or1-ethyl-3(4-azonia-4,4-dimetholpentyl) carbodiimide. Aspartyl orglutamyl can also be converted to asparaginyl and glutaminyl residues byreaction with ammonium ions.

Mimetics of basic amino acids can be generated by substitution with,e.g., (in addition to lysine and arginine) the amino acids ornithine,citrulline, or (guanidino)-acetic acid, or (guanidino)alkyl-acetic acid,where alkyl is defined above. Nitrile derivative (e.g., containing theCN-moiety in place of COOH) can be substituted for asparagine orglutamine. Asparaginyl and glutaminyl residues can be deaminated to thecorresponding aspartyl or glutamyl residues.

Arginine residue mimetics can be generated by reacting arginyl with,e.g., one or more conventional reagents, including, e.g., phenylglyoxal,2,3-butanedione, 1,2-cyclohexanedione, or ninhydrin, preferably underalkaline conditions.

Tyrosine residue mimetics can be generated by reacting tyrosyl with,e.g., aromatic diazonium compounds or tetranitromethane.N-acetylimidizol and tetranitromethane can be used to form O-acetyltyrosyl species and 3-nitro derivatives, respectively.

Cysteine residue mimetics can be generated by reacting cysteinylresidues with, e.g., alpha-haloacetates such as 2-chloroacetic acid orchloroacetamide and corresponding amines, to give carboxymethyl orcarboxyamidomethyl derivatives. Cysteine residue mimetics can also begenerated by reacting cysteinyl residues with, e.g.,bromo-trifluoroacetone, alpha-bromo-beta-(5-imidozoyl) propionic acid;chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide;methyl 2-pyridyl disulfide; p-chloromercuribenzoate; 2-chloromercuri-4nitrophenol; or, chloro-7-nitrobenzo-oxa-1,3-diazole.

Lysine mimetics can be generated (and amino terminal residues can bealtered) by reacting lysinyl with, e.g., succinic or other carboxylicacid anhydrides. Lysine and other alpha-amino-containing residuemimetics can also be generated by reaction with imidoesters, such asmethyl picolinimidate, pyridoxal phosphate, pyridoxal,chloroborohydride, trinitrobenzenesulfonic acid, O-methylisourea, 2,4,pentanedione, and transamidase-catalyzed reactions with glyoxylate.

Mimetics of methionine can be generated by reaction with, e.g.,methionine sulfoxide. Mimetics of proline include, e.g., pipecolic acid,thiazolidine carboxylic acid, 3- or 4-hydroxy proline, dehydroproline,3- or 4-methylproline, or 3,3,-dimethylproline. Histidine residuemimetics can be generated by reacting histidyl with, e.g.,diethylprocarbonate or para-bromophenacyl bromide.

Other mimetics include, e.g., those generated by hydroxylation ofproline and lysine; phosphorylation of the hydroxyl groups of seryl orthreonyl residues; methylation of the alpha-amino groups of lysine,arginine and histidine; acetylation of the N-terminal amine; methylationof main chain amide residues or substitution with N-methyl amino acids;or amidation of C-terminal carboxyl groups.

A component of a natural polypeptide (e.g., a PL polypeptide or PDZpolypeptide) can also be replaced by an amino acid (or peptidomimeticresidue) of the opposite chirality. Thus, any amino acid naturallyoccurring in the L-configuration (which can also be referred to as the Ror S, depending upon the structure of the chemical entity) can bereplaced with the amino acid of the same chemical structural type or apeptidomimetic, but of the opposite chirality, generally referred to asthe D-amino acid, but which can additionally be referred to as the R- orS-form.

The mimetics of the invention can also include compositions that containa structural mimetic residue, particularly a residue that induces ormimics secondary structures, such as a beta turn, beta sheet, alphahelix structures, gamma turns, and the like. For example, substitutionof natural amino acid residues with D-amino acids; N-alpha-methyl aminoacids; C-alpha-methyl amino acids; or dehydroamino acids within apeptide can induce or stabilize beta turns, gamma turns, beta sheets oralpha helix conformations. Beta turn mimetic structures have beendescribed, e.g., by Nagai (1985) Tet. Lett. 26:647-650; Feigl (1986) J.Amer. Chem. Soc. 108:181-182; Kahn (1988) J. Amer. Chem. Soc.110:1638-1639; Kemp (1988) Tet. Lett. 29:5057-5060; Kahn (1988) J.Molec. Recognition 1:75-79. Beta sheet mimetic structures have beendescribed, e.g., by Smith (1992) J. Amer. Chem. Soc. 114:10672-10674.For example, a type VI beta turn induced by a cis amide surrogate,1,5-disubstituted tetrazol, is described by Beusen (1995) Biopolymers36:181-200. Incorporation of achiral omega-amino acid residues togenerate polymethylene units as a substitution for amide bonds isdescribed by Banerjee (1996) Biopolymers 39:769-777. Secondarystructures of polypeptides can be analyzed by, e.g., high-field 1H NMRor 2D NMR spectroscopy, see, e.g., Higgins (1997) J. Pept. Res.50:421-435. See also, Hruby (1997) Biopolymers 43:219-266, Balaji, etal., U.S. Pat. No. 5,612,895.

As used herein, “peptide variants” and “conservative amino acidsubstitutions” refer to peptides that differ from a reference peptide(e.g., a peptide having the sequence of the carboxy-terminus of aspecified PL protein) by substitution of an amino acid residue havingsimilar properties (based on size, polarity, hydrophobicity, and thelike). Thus, insofar as the compounds that are encompassed within thescope of the invention are partially defined in terms of amino acidresidues of designated classes, the amino acids may be generallycategorized into three main classes: hydrophilic amino acids,hydrophobic amino acids and cysteine-like amino acids, dependingprimarily on the characteristics of the amino acid side chain. Thesemain classes may be further divided into subclasses. Hydrophilic aminoacids include amino acids having acidic, basic or polar side chains andhydrophobic amino acids include amino acids having aromatic or apolarside chains. Apolar amino acids may be further subdivided to include,among others, aliphatic amino acids. The definitions of the classes ofamino acids as used herein are as follows:

“Hydrophobic Amino Acid” refers to an amino acid having a side chainthat is uncharged at physiological pH and that is repelled by aqueoussolution. Examples of genetically encoded hydrophobic amino acidsinclude Ile, Leu and Val. Examples of non-genetically encodedhydrophobic amino acids include t-BuA.

“Aromatic Amino Acid” refers to a hydrophobic amino acid having a sidechain containing at least one ring having a conjugated π-electron system(aromatic group). The aromatic group may be further substituted withgroups such as alkyl, alkenyl, alkynyl, hydroxyl, sulfanyl, nitro andamino groups, as well as others. Examples of genetically encodedaromatic amino acids include Phe, Tyr and Trp. Commonly encounterednon-genetically encoded aromatic amino acids include phenylglycine,2-naphthylalanine, β-2-thienylalanine,1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid,4-chloro-phenylalanine, 2-fluorophenyl-alanine, 3-fluorophenylalanineand 4-fluorophenylalanine.

“Apolar Amino Acid” refers to a hydrophobic amino acid having a sidechain that is generally uncharged at physiological pH and that is notpolar. Examples of genetically encoded apolar amino acids include Gly,Pro and Met. Examples of non-encoded apolar amino acids include Cha.

“Aliphatic Amino Acid” refers to an apolar amino acid having a saturatedor unsaturated straight chain, branched or cyclic hydrocarbon sidechain. Examples of genetically encoded aliphatic amino acids includeAla, Leu, Val and Ile. Examples of non-encoded aliphatic amino acidsinclude Nle.

“Hydrophilic Amino Acid” refers to an amino acid having a side chainthat is attracted by aqueous solution. Examples of genetically encodedhydrophilic amino acids include Ser and Lys. Examples of non-encodedhydrophilic amino acids include Cit and hCys.

“Acidic Amino Acid” refers to a hydrophilic amino acid having a sidechain pK value of less than 7. Acidic amino acids typically havenegatively charged side chains at physiological pH due to loss of ahydrogen ion. Examples of genetically encoded acidic amino acids includeAsp and Glu.

“Basic Amino Acid” refers to a hydrophilic amino acid having a sidechain pK value of greater than 7. Basic amino acids typically havepositively charged side chains at physiological pH due to associationwith hydronium ion. Examples of genetically encoded basic amino acidsinclude Arg, Lys and His. Examples of non-genetically encoded basicamino acids include the non-cyclic amino acids ornithine,2,3-diaminopropionic acid, 2,4-diaminobutyric acid and homoarginine.

“Polar Amino Acid” refers to a hydrophilic amino acid having a sidechain that is uncharged at physiological pH, but which has a bond inwhich the pair of electrons shared in common by two atoms is held moreclosely by one of the atoms. Examples of genetically encoded polar aminoacids include Asx and Glx. Examples of non-genetically encoded polaramino acids include citrulline, N-acetyl lysine and methioninesulfoxide.

“Cysteine-Like Amino Acid” refers to an amino acid having a side chaincapable of forming a covalent linkage with a side chain of another aminoacid residue, such as a disulfide linkage. Typically, cysteine-likeamino acids generally have a side chain containing at least one thiol(SH) group. Examples of genetically encoded cysteine-like amino acidsinclude Cys. Examples of non-genetically encoded cysteine-like aminoacids include homocysteine and penicillamine.

As will be appreciated by those having skill in the art, the aboveclassification are not absolute—several amino acids exhibit more thanone characteristic property, and can therefore be included in more thanone category. For example, tyrosine has both an aromatic ring and apolar hydroxyl group. Thus, tyrosine has dual properties and can beincluded in both the aromatic and polar categories. Similarly, inaddition to being able to form disulfide linkages, cysteine also hasapolar character. Thus, while not strictly classified as a hydrophobicor apolar amino acid, in many instances cysteine can be used to conferhydrophobicity to a peptide.

Certain commonly encountered amino acids which are not geneticallyencoded of which the peptides and peptide analogues of the invention maybe composed include, but are not limited to, β-alanine (b-Ala) and otheromega-amino acids such as 3-aminopropionic acid (Dap),2,3-diaminopropionic acid (Dpr), 4-aminobutyric acid and so forth;α-aminoisobutyric acid (Aib); ε-aminohexanoic acid (Aha); δ-aminovalericacid (Ava); N-methylglycine or sarcosine (MeGly); ornithine (Om);citrulline (Cit); t-butylalanine (t-BuA); t-butylglycine (t-BuG);N-methylisoleucine (MeIle); phenylglycine (Phg); cyclohexylalanine(Cha); norleucine (Nle); 2-naphthylalanine (2-Nal);4-chlorophenylalanine (Phe(4-Cl)); 2-fluorophenylalanine (Phe(2-F));3-fluorophenylalanine (Phe(3-F)); 4-fluorophenylalanine (Phe(4-F));penicillamine (Pen); 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid(Tic); P-2-thienylalanine (Thi); methionine sulfoxide (MSO);homoarginine (hArg); N-acetyl lysine (AcLys); 2,3-diaminobutyric acid(Dab); 2,3-diaminobutyric acid (Dbu); p-aminophenylalanine (Phe(pNH₂));N-methyl valine (MeVal); homocysteine (hCys) and homoserine (hSer).These amino acids also fall conveniently into the categories definedabove.

The classifications of the above-described genetically encoded andnon-encoded amino acids are summarized in TABLE 1, below. It is to beunderstood that TABLE 1 is for illustrative purposes only and does notpurport to be an exhaustive list of amino acid residues which maycomprise the peptides and peptide analogues described herein. Otheramino acid residues which are useful for making the peptides and peptideanalogues described herein can be found, e.g., in Fasman, 1989, CRCPractical Handbook of Biochemistry and Molecular Biology, CRC Press,Inc., and the references cited therein. Amino acids not specificallymentioned herein can be conveniently classified into the above-describedcategories on the basis of known behavior and/or their characteristicchemical and/or physical properties as compared with amino acidsspecifically identified.

TABLE 1 Genetically Classification Encoded Genetically Non-EncodedHydrophobic Aromatic F, Y, W Phg, Nal, Thi, Tic, Phe(4-Cl), Phe(2-F),Phe(3-F), Phe(4-F), Pyridyl Ala, Benzothienyl Ala Apolar M, G, PAliphatic A, V, L, I t-BuA, t-BuG, MeIle, Nle, MeVal, Cha, bAla, MeGly,Aib Hydrophilic Acidic D, E Basic H, K, R Dpr, Orn, hArg, Phe(p-NH₂),DBU, A₂BU Polar Q, N, S, T, Y Cit, AcLys, MSO, hSer Cysteine-Like C Pen,hCys, p-methyl Cys

In the case of the PDZ domains described herein, a “HPV E6-bindingvariant” of a particular PDZ domain is a PDZ domain variant that retainsHPV E6 PDZ ligand binding activity. Assays for determining whether a PDZdomain variant binds HPV E6 are described in great detail below, andguidance for identifying which amino acids to change in a specific PDZdomain to make it into a variant may be found in a variety of sources.In one example, a PDZ domain may be compared to other PDZ domainsdescribed herein and amino acids at corresponding positions may besubstituted, for example. In another example, the sequence a PDZ domainof a particular PDZ protein may be compared to the sequence of anequivalent PDZ domain in an equivalent PDZ protein from another species.For example, the sequence a PDZ domain from a human PDZ protein may becompared to the sequence of other known and equivalent PDZ domains fromother species (e.g., mouse, rat, etc.) and any amino acids that arevariant between the two sequences may be substituted into the human PDZdomain to make a variant of the PDZ domain. For example, the sequence ofthe human MAGI-1 PDZ domain 2 may be compared to equivalent MAGI-1 PDZdomains from other species (e.g. mouse Genbank gi numbers 7513782 and28526157 or other homologous sequences) to identify amino acids that maybe substituted into the human MAGI-1-PDZ domain to make a variantthereof. Such method may be applied to any of the MAGI-1 PDZ domainsdescribed herein. Minimal MAGI-PDZ domain 2 sequence is provided as SEQID NOS:293-301. Particular variants may have 1, up to 5, up to about 10,up to about 15, up to about 20 or up to about 30 or more, usually up toabout 50 amino acid changes as compared to a sequence set forth in thesequence listing. Exemplary MAGI-1 PDZ variants include the sequencesset forth in SEQ ID NOS: 302-330. In making a variant, if a GFG motif ispresent in a PDZ domain, in general, it should not be altered insequence.

In general, variant PDZ domain polypeptides have a PDZ domain that hasat least about 70 or 80%, usually at least about 90%, and more usuallyat least about 98% sequence identity with a variant PDZ domainpolypeptide described herein, as measured by BLAST 2.0 using defaultparameters, over a region extending over the entire PDZ domain.

As used herein, a “detectable label” has the ordinary meaning in the artand refers to an atom (e.g., radionuclide), molecule (e.g.,fluorescein), or complex, that is or can be used to detect (e.g., due toa physical or chemical property), indicate the presence of a molecule orto enable binding of another molecule to which it is covalently bound orotherwise associated. The term “label” also refers to covalently boundor otherwise associated molecules (e.g., a biomolecule such as anenzyme) that act on a substrate to produce a detectable atom, moleculeor complex. Detectable labels suitable for use in the present inventioninclude any composition detectable by spectroscopic, photochemical,biochemical, immunochemical, electrical, optical or chemical means.Labels useful in the present invention include biotin for staining withlabeled streptavidin conjugate, magnetic beads (e.g., Dynabeads™),fluorescent dyes (e.g., fluorescein, Texas red, rhodamine, greenfluorescent protein, enhanced green fluorescent protein, and the like),radiolabels (e.g., ³H, ¹²⁵I, ³⁵S, ¹⁴C, or ³²P), enzymes (e.g.,hydrolases, particularly phosphatases such as alkaline phosphatase,esterases and glycosidases, or oxidoreductases, particularly peroxidasessuch as horse radish peroxidase, and others commonly used in ELISAs),substrates, cofactors, inhibitors, chemiluminescent groups, chromogenicagents, and colorimetric labels such as colloidal gold or colored glassor plastic (e.g., polystyrene, polypropylene, latex, etc.) beads.Patents teaching the use of such labels include U.S. Pat. Nos.3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and4,366,241. Means of detecting such labels are well known to those ofskill in the art. Thus, for example, radiolabels and chemiluminescentlabels may be detected using photographic film or scintillationcounters, fluorescent markers may be detected using a photodetector todetect emitted light (e.g., as in fluorescence-activated cell sorting).Enzymatic labels are typically detected by providing the enzyme with asubstrate and detecting the reaction product produced by the action ofthe enzyme on the substrate, and colorimetric labels are detected bysimply visualizing the colored label. Thus, a label is any compositiondetectable by spectroscopic, photochemical, biochemical, immunochemical,electrical, optical or chemical means. The label may be coupled directlyor indirectly to the desired component of the assay according to methodswell known in the art. Non-radioactive labels are often attached byindirect means. Generally, a ligand molecule (e.g., biotin) iscovalently bound to the molecule. The ligand then binds to ananti-ligand (e.g., streptavidin) molecule which is either inherentlydetectable or covalently bound to a signal generating system, such as adetectable enzyme, a fluorescent compound, or a chemiluminescentcompound. A number of ligands and anti-ligands can be used. Where aligand has a natural anti-ligand, for example, biotin, thyroxine, andcortisol, it can be used in conjunction with the labeled, naturallyoccurring anti-ligands. Alternatively, any haptenic or antigeniccompound can be used in combination with an antibody. The molecules canalso be conjugated directly to signal generating compounds, e.g., byconjugation with an enzyme or fluorophore. Means of detecting labels arewell known to those of skill in the art. Thus, for example, where thelabel is a radioactive label, means for detection include ascintillation counter, photographic film as in autoradiography, orstorage phosphor imaging. Where the label is a fluorescent label, it maybe detected by exciting the fluorochrome with the appropriate wavelengthof light and detecting the resulting fluorescence. The fluorescence maybe detected visually, by means of photographic film, by the use ofelectronic detectors such as charge coupled devices (CCDs) orphotomultipliers and the like. Similarly, enzymatic labels may bedetected by providing the appropriate substrates for the enzyme anddetecting the resulting reaction product. Also, simple colorimetriclabels may be detected by observing the color associated with the label.It will be appreciated that when pairs of fluorophores are used in anassay, it is often preferred that they have distinct emission patterns(wavelengths) so that they can be easily distinguished.

As used herein, the term “substantially identical” in the context ofcomparing amino acid sequences, means that the sequences have at leastabout 70%, at least about 80%, or at least about 90% amino acid residueidentity when compared and aligned for maximum correspondence. Analgorithm that is suitable for determining percent sequence identity andsequence similarity is the FASTA algorithm, which is described inPearson, W. R. & Lipman, D. J., 1988, Proc. Natl. Acad. Sci. U.S.A. 85:2444. See also W. R. Pearson, 1996, Methods Enzymol. 266: 227-258.Preferred parameters used in a FASTA alignment of DNA sequences tocalculate percent identity are optimized, BL50 Matrix 15: −5, k-tuple=2;joining penalty=40, optimization=28; gap penalty−12, gap lengthpenalty=−2; and width=16.

As used herein, the terms “sandwich”, “sandwich ELISA”, “Sandwichdiagnostic” and “capture ELISA” all refer to the concept of detecting abiological polypeptide with two different test agents. For example, aPDZ protein could be directly or indirectly attached to a solid support.Test sample could be passed over the surface and the PDZ protein couldbind it's cognate PL protein(s). A labeled antibody or alternativedetection reagent could then be used to determine whether a specific PLprotein had bound the PDZ protein.

By “solid phase support” or “carrier” is intended any support capable ofbinding polypeptide, antigen or antibody. Well-known supports orcarriers, include glass, polystyrene, polypropylene, polyethylene,dextran, nylon, amylases, natural and modified celluloses,polyacrylamides, agaroses, and magnetite. The nature of the carrier canbe either soluble to some extent or insoluble for the purposes of thepresent invention. The support material can have virtually any possiblestructural configuration so long as the coupled molecule is capable ofbinding to a PDZ domain polypeptide or an E6 antibody. Thus, the supportconfiguration can be spherical, as in a bead, or cylindrical, as in theinside surface of a test tube, or the external surface of a rod.Alternatively, the surface can be flat, such as a sheet, culture dish,test strip, etc. Those skilled in the art will know many other suitablecarriers for binding antibody, peptide or antigen, or can ascertain thesame by routine experimentation.

As used herein, the terms “test compound” or “test agent” are usedinterchangeably and refer to a candidate agent that may haveenhancer/agonist, or inhibitor/antagonist activity, e.g., inhibiting orenhancing an interaction such as PDZ-PL binding. The candidate agents ortest compounds may be any of a large variety of compounds, bothnaturally occurring and synthetic, organic and inorganic, and includingpolymers (e.g., oligopeptides, polypeptides, oligonucleotides, andpolynucleotides), small molecules, antibodies (as broadly definedherein), sugars, fatty acids, nucleotides and nucleotide analogs,analogs of naturally occurring structures (e.g., peptide mimetics,nucleic acid analogs, and the like), and numerous other compounds. Incertain embodiment, test agents are prepared from diversity libraries,such as random or combinatorial peptide or non-peptide libraries. Manylibraries are known in the art that can be used, e.g., chemicallysynthesized libraries, recombinant (e.g., phage display libraries), andin vitro translation-based libraries. Examples of chemically synthesizedlibraries are described in Fodor et al., 1991, Science 251:767-773;Houghten et al., 1991, Nature 354:84-86; Lam et al., 1991, Nature354:82-84; Medynski, 1994, Bio/Technology 12:709-710; Gallop et al.,1994, J. Medicinal Chemistry 37(9):1233-1251; Ohlmeyer et al., 1993,Proc. Natl. Acad. Sci. USA 90:10922-10926; Erb et al., 1994, Proc. Natl.Acad. Sci. USA 91:11422-11426; Houghten et al., 1992, Biotechniques13:412; Jayawickreme et al., 1994, Proc. Natl. Acad. Sci. USA91:1614-1618; Salmon et al., 1993, Proc. Natl. Acad. Sci. USA90:11708-11712; PCT Publication No. WO 93/20242; and Brenner and Lerner,1992, Proc. Natl. Acad. Sci. USA 89:5381-5383. Examples of phage displaylibraries are described in Scott and Smith, 1990, Science 249:386-390;Devlin et. al., 1990, Science, 249:404-406; Christian, R. B., et al.,1992, J. Mol. Biol. 227:711-718); Lenstra, 1992, J. Immunol. Meth.152:149-157; Kay et al., 1993, Gene 128:59-65; and PCT Publication No.WO 94/18318 dated Aug. 18, 1994. In vitro translation-based librariesinclude but are not limited to those described in PCT Publication No. WO91/05058 dated Apr. 18, 1991; and Mattheakis et al., 1994, Proc. Natl.Acad. Sci. USA 91:9022-9026. By way of examples of nonpeptide libraries,a benzodiazepine library (see e.g., Bunin et al., 1994, Proc. Natl.Acad. Sci. USA 91:4708-4712) can be adapted for use. Peptoid libraries(Simon et al., 1992, Proc. Natl. Acad. Sci. USA 89:9367-9371) can alsobe used. Another example of a library that can be used, in which theamide functionalities in peptides have been permethylated to generate achemically transformed combinatorial library, is described by Ostresh etal. (1994, Proc. Natl. Acad. Sci. USA 91:11138-11142).

The term “specific binding” refers to binding between two molecules, forexample, a ligand and a receptor, characterized by the ability of amolecule (ligand) to associate with another specific molecule (receptor)even in the presence of many other diverse molecules, i.e., to showpreferential binding of one molecule for another in a heterogeneousmixture of molecules. Specific binding of a ligand to a receptor is alsoevidenced by reduced binding of a detectably labeled ligand to thereceptor in the presence of excess unlabeled ligand (i.e., a bindingcompetition assay).

In some embodiments “proteasome inhibitors”, i.e., inhibitors of theproteasome, may be used. These inhibitors, includingcarbobenzoxyl-leucinyl-leuciny-1 norvalinal II (MG 115) or CBZ-LLL canbe purchased from chemical supply companies (e.g., Sigma). As a skilledperson would understand, proteasome inhibitors are not proteaseinhibitors.

As used herein, a “plurality” of PDZ proteins (or corresponding PDZdomains or PDZ fusion polypeptides) has its usual meaning. In someembodiments, the plurality is at least 5, and often at least 25, atleast 40, or at least 60 different PDZ proteins. In some embodiments,the plurality is selected from the list of PDZ polypeptides listed inTABLE 2. In some embodiments, the plurality of different PDZ proteinsare from (i.e., expressed in) a particular specified tissue or aparticular class or type of cell. In some embodiments, the plurality ofdifferent PDZ proteins represents a substantial fraction (e.g.,typically at least 50%, more often at least 80%) of all of the PDZproteins known to be, or suspected of being, expressed in the tissue orcell(s), e.g., all of the PDZ proteins known to be present inlymphocytes or hematopoetic cells. In some embodiments, the plurality isat least 50%, usually at least 80%, at least 90% or all of the PDZproteins disclosed herein as being expressed in a particular cell.

When referring to PL peptides (or the corresponding proteins, e.g.,corresponding to those listed in TABLE 3, or elsewhere herein) a“plurality” may refer to at least 5, at least 10, and often at least 16PLs such as those specifically listed herein, or to the classes andpercentages set forth supra for PDZ domains.

II. Overview

The present inventors have identified a large number of interactionsbetween PDZ proteins and PL proteins that can play a significant role inthe biological function of a variety of physiological systems. As usedherein, the term “biological function” in the context of a cell, refersto a detectable biological activity normally carried out by the cell,e.g., a phenotypic change such as cell proliferation (e.g., cancer),cell activation, cytokine release, degranulation, tyrosinephosphorylation, ion (e.g., calcium) flux, metabolic activity,apoptosis, changes in gene expression, maintenance of cell structure,cell migration, adherence to a substrate, signal transduction, cell-cellinteractions, and others described herein or known in the art.

Because the interactions involve proteins that are involved in diversephysiological systems (see Background section supra), the methodsprovided herein can be utilized to broadly or selectively diagnoseinappropriate cellular phenotypes or pathogenic infections. Methods arealso disclosed herein for determining whether vertebrate biologicalsamples contain pathogenic organisms using PDZ:PL protein interactions.

As will be discussed in great detail below, the use of PDZ-PLinteractions for diagnostic purposes is amenable to a number ofdifferent test formats and is not intended to be limited by thediscussion herein. Diagnostic tests could be formatted for ELISA assays,as a dipstick test such as is used for pregnancy tests, as a film testthat can be incubated with test sample, as a slide test that samplecould be placed upon, or other such mediums. Such formats are well knownin the art, and are described in U.S. Pat. Nos. 6,180,417, 4,703,0175,591,645

III. PDZ Protein and PL Protein Interactions

TABLE 4 lists PDZ proteins and PL proteins which the current inventorshave identified as binding to one another. Each page of TABLE 4 includesfour columns. The columns in each section are number from left to rightsuch that the left-most column in each section is column 1 and theright-most column in each section is column 4. Thus, the first column ineach section is labeled “HPV Strain” and lists the various E6 proteinsthat contain the PDZ-Ligand sequences (PLs) that were examined (shown inparenthesis). This column lists C-terminal four amino acids thatcorrespond to the carboxyl-terminal end of a 20 amino acid peptide usedin this binding study. All ligands are biotinylated at theamino-terminus and partial sequences are presented in TABLE 3.

The PDZ protein (or proteins) that interact(s) with HPV E6-PL peptidesare listed in the second column labeled “PDZ binding partner”. Thiscolumn provides the gene name for the PDZ portion of the GST-PDZ fusionthat interacts with the PDZ-ligand to the left. For PDZdomain-containing proteins with multiple domains the domain number islisted to the right of the PDZ (i.e., in column 4 labeled “PDZ Domain”),and indicates the PDZ domain number when numbered from theamino-terminus to the carboxy-terminus. This table only listsinteractions of a stronger nature, e.g., those that give a ‘4’ or ‘5’classification in the ‘G assay’. “Classification” is a measure of thelevel of binding. In particular, it provides an absorbance value at 450m which indicates the amount of PL peptide bound to the PDZ protein. Thefollowing numerical values have the following meanings: ‘1’—A₄₅₀ nm 0-1;‘2’—A₄₅₀ nm 1-2; ‘3’—A₄₅₀ nm 2-3; ‘4’—A₄₅₀ nm 3-4; ‘5’—A₄₅₀ nm of 4 morethan 2× repeated; ‘0’—A₄₅₀ nm 0, i.e., not found to interact.

The third and fourth columns of TABLE 4 are merely a repetition of thecolumns 1 and 2 with different E6 PLs tested and the PDZs bound by themat higher affinity.

Further information regarding these PL proteins and PDZ proteins isprovided in TABLES 2 and 3 and EXAMPLEs 4 and 5. In particular, TABLE 3provides a listing of the partial amino acid sequences of peptides usedin the assays. When numbered from left to right, the first columnlabeled “HPV strain” provides the HPV strain number used to refer to theE6 protein from that strain. The column labeled “E6 C-terminal sequence”provides the predicted sequence of the carboxy-terminal 10 amino acidsof the E6 protein. The third column labeled “PL yes/no” designateswhether the E6-PL sequence contains sequence elements predicted to bindto PDZ domains. The final column labeled “oncogenic” indicates that thisHPV strain is known to cause cervical cancer as determined by theNational Cancer Institute (NCI, 2001).

EXAMPLE 5 lists representative sequences of PDZ domains cloned into avector (PGEX-3× vector) for production of GST-PDZ fusion proteins(Pharmacia). An extended list of PDZ domains cloned into pGEX vectorsfor production of GST-PDZ fusion proteins is listed in U.S. patent Ser.No. 09/724,553.

As discussed in detail herein, the PDZ proteins listed in TABLE 2 arenaturally occurring proteins containing a PDZ domain. Only significantinteractions are presented in this table. Thus, the present invention isparticularly directed to the detection and modulation of interactionsbetween a PDZ protein and PL protein. In a similar manner, PDZ domainsthat bind other pathogens can be used to diagnose infection. Additionalexamples of PL proteins from pathogens suitable for diagnosticapplications are included in TABLE 8, but are not intended to limit thescope of the invention.

In another embodiment of the invention, cellular abnormalities ordiseases can be diagnosed through the detection of imbalances in theexpression levels of cellular PDZ proteins or PL proteins. Using eitherthe PL protein or the PDZ protein in an assay derived from the ‘A assay’or ‘G assay’ one can determine the protein expression levels of bindingpartners in a normal or abnormal cell. Differences in protein expressionlevels have been correlated with a number of diseases.

In certain embodiments of the invention, a PDZ protein is used todiagnose the presence of a PL protein from a pathogenic organism.Examples of pathogenic organisms with PL sequences include, but are notlimited to, viruses such as Human Papillomaviruses, Hepatitus B virus,Adenovirus, Human T Cell Leukemia Virus, bacteria and fungi.

IV. Assays for Detection of PDZ Proteins or PDZ-Ligand Proteins (PLProteins)

Two complementary assays, termed “A” and “G”, were developed to detectbinding between a PDZ-domain polypeptide and candidate PDZ ligand. Ineach of the two different assays, binding is detected between a peptidehaving a sequence corresponding to the C-terminus of a proteinanticipated to bind to one or more PDZ domains (i.e. a candidate PLpeptide) and a PDZ-domain polypeptide (typically a fusion proteincontaining a PDZ domain). In the “A” assay, the candidate PL peptide isimmobilized and binding of a soluble PDZ-domain polypeptide to theimmobilized peptide is detected (the “A” assay is named for the factthat in one embodiment an avidin surface is used to immobilize thepeptide). In the “G” assay, the PDZ-domain polypeptide is immobilizedand binding of a soluble PL peptide is detected (The “G” assay is namedfor the fact that in one embodiment a GST-binding surface is used toimmobilize the PDZ-domain polypeptide). Preferred embodiments of theseassays are described in detail infra. However, it will be appreciated byordinarily skilled practitioners that these assays can be modified innumerous ways while remaining useful for the purposes of the presentinvention.

A. Production of Fusion Proteins Containing PDZ-Domains

GST-PDZ domain fusion proteins were prepared for use in the assays ofthe invention. PCR products containing PDZ encoding domains (asdescribed supra) were subcloned into an expression vector to permitexpression of fusion proteins containing a PDZ domain and a heterologousdomain (i.e., a glutathione-S transferase sequence, “GST”). PCR products(i.e., DNA fragments) representing PDZ domain encoding DNA wereextracted from agarose gels using the “Sephaglas” gel extraction system(Pharmacia) according to the manufacturer's recommendations.

As noted supra, PCR primers were designed to include endonucleaserestriction sites to facilitate ligation of PCR fragments into a GSTgene fusion vector (pGEX-3×; Pharmacia, GenBank accession no. XXU13852)in-frame with the glutathione-S transferase coding sequence. This vectorcontains an IPTG inducible lacZ promoter. The pGEX-3× vector waslinearized using Bam HI and Eco RI or, in some cases, Eco RI or Sma I,and dephosphorylated. For most cloning approaches, double digestion withBam HI and Eco RI was performed, so that the ends of the PCR fragmentsto clone were Bam HI and Eco RI. In some cases, restriction endonucleasecombinations used were Bgl II and Eco RI, Bam HI and Mfe I, or Eco RIonly, Sma I only, or BamHI only. When more than one PDZ domain wascloned, the DNA portion cloned represents the PDZ domains and the cDNAportion located between individual domains. Precise locations of clonedfragments used in the assays are indicated in U.S. patent application(60/360,061). DNA linker sequences between the GST portion and the PDZdomain containing DNA portion vary slightly, dependent on which of theabove described cloning sites and approaches were used. As aconsequence, the amino acid sequence of the GST-PDZ fusion proteinvaries in the linker region between GST and PDZ domain. Protein linkersequences corresponding to different cloning sites/approaches are shownbelow. Linker sequences (vector DNA encoded) are bold, PDZ domaincontaining gene derived sequences are in italics.

1) GST-BamHI/BamHI-PDZ domain insert

Gly-Ile-PDZ domain insert

2) GST-BamHI/BglII-PDZ domain insert

Gly-Ile-PDZ domain insert

3) GST-EcoRI/EcoRI-PDZ domain insert

Gly-Ile-Pro-Gly-Asn-PDZ domain insert

4) GST-SmaI/SmaI-PDZ domain insert

Gly-Ile-Pro-PDZ domain insert

The PDZ-encoding PCR fragment and linearized pGEX-3× vector were ethanolprecipitated and resuspended in 10 ul standard ligation buffer. Ligationwas performed for 4-10 hours at 7° C. using T4 DNA ligase. It will beunderstood that some of the resulting constructs include very shortlinker sequences and that, when multiple PDZ domains were cloned, theconstructs included some DNA located between individual PDZ domains.

The ligation products were transformed in DH5alpha or BL-21 E. colibacteria strains. Colonies were screened for presence and identity ofthe cloned PDZ domain containing DNA as well as for correct fusion withthe glutathione S-transferase encoding DNA portion by PCR and bysequence analysis. Positive clones were tested in a small-scale assayfor expression of the GST/PDZ domain fusion protein and, if expressing,these clones were subsequently grown up for large scale preparations ofGST/PDZ fusion protein.

GST-PDZ domain fusion protein was overexpressed following addition ofIPTG to the culture medium and purified. Detailed procedure of smallscale and large-scale fusion protein expression and purification aredescribed in “GST Gene Fusion System” (second edition, revision 2;published by Pharmacia). In brief, a small culture (50 mls) containing abacterial strain (DH5α, BL21 or JM109) with the fusion protein constructwas grown overnight in 2×YT media at 37° C. with the appropriateantibiotic selection (100 ug/ml ampicillin; a.k.a. 2×YT-amp). Theovernight culture was poured into a fresh preparation of 2×YT-amp(typically 1 liter) and grown until the optical density (OD) of theculture was between 0.5 and 0.9 (approximately 2.5 hours). IPTG(isopropyl β-D-thiogalactopyranoside) was added to a final concentrationof 1.0 mM to induce production of GST fusion protein, and culture wasgrown an additional 1 hour. All following steps, includingcentrifugation, were performed on ice or at 4° C. Bacteria werecollected by centrifugation (4500×g) and resuspended in Buffer A− (50 mMTris, pH 8.0, 50 mM dextrose, 1 mM EDTA, 200 uMphenylmethylsulfonylfluoride). An equal volume of Buffer A+ (Buffer A−,4 mg/ml lysozyme) was added and incubated on ice for 3 min to lysebacteria, or until lysis had begun. An equal volume of Buffer B (10 mMTris, pH 8.0, 50 mM KCl, 1 mM EDTA. 0.5% Tween-20, 0.5% NP40 (a.k.a.IGEPAL CA-630), 200 uM phenylmethylsulfonylfluoride) was added andincubated for an additional 20 min on ice. The bacterial cell lysate wascentrifuged (×20,000 g), and supernatant was run over a columncontaining 20 ml Sepharose CL-4B (Pharmacia) “precolumn beads,” i.e.,sepharose beads without conjugated glutathione that had been previouslywashed with 3 bed volumes PBS. The flow-through was added to glutathioneSepharose 4B (Pharmacia, cat no. 17-0765-01) previously swelled(rehydrated) in 1× phosphate-buffered saline (PBS) and incubated whilerotating for 30 min-1 hr. The supernatant-Sepharose slurry was pouredinto a column and washed with at least 20 bed volumes of 1×PBS. GSTfusion protein was eluted off the glutathione sepharose by applying0.5-1.0 ml aliquots of 5 mM glutathione and collected as separatefractions. Concentrations of fractions were determined by readingabsorbance at 280 nm and calculating concentration using the absorbanceand extinction coefficient. Those fractions containing the highestconcentration of fusion protein were pooled and an equal) volume of 70%glycerol was added to a final concentration of 35% glycerol. Fusionproteins were assayed for size and quality by SDS gel electrophoresis(PAGE) as described in “Sambrook.” Fusion protein aliquots were storedat minus 80° C. and at minus 20° C.

TABLE 2 PDZ Domains Used in Assays of the Invention Gene Name GI or Acc#PDZ# Sequence fused to GST Construct Seq ID 26s subunit 9184389 1RDMAEAHKEAMSRKLGQSESQGPPRAFAKVNSISPGSPSIAGLQVDDEIVEFGSVNTQNFQAS 1 p27LHNIGSVVQHSEGALAPTILLSVSM AF6 430993 1LRKEPEIITVTLKKQNGMGLSIVAAKGAGQDKLGIYVKSVVKGGAADVDGRLAAGDQLLSVDGR 2SLVGLSQERAAELMTRTSSVVTLEVAKQG AIPC 12751451 1LIRPSVISIIGLYKEKGKGLGFSIAGGRDCIRGQMGIFVKTIFPNGSAAEDGRLKEGDEILDVN 3GIPIKGLTFQEAIHTFKQIRSGLFVLTVRTKLVSPSLTNSS AIPC 12751451 2GISSLGRKTPGPKDRIVMEVTLNKEPRVGLGIGACCLALENSPPGIYIHSLAPGSVAKMESNLS 4RGDQILEVNSVNVRHAALSKVHAILSKCPPGPVRLVIGRHPNPKVSEQEMDEVIARSTYQESK EANSSAIPC 12751451 3QSENEEDVCFIVLNRKEGSGLGFSVAGGTDVEPKSITVHRVFSQGAASQEGTMNRGDFLLSVNG 5ASLAGLAHGNVLKVLHQAQLHKDALVVIKKGMDQPRPSNSS AIPC 12751451 4LGRSVAVHDALCVEVLKTSAGLGLSLDGGKSSVTGDGPLVIKRVYKGGAAEQAGIIEAGDEIL 6AINGKPLVGLMHFDAWNIMKSVPEGPVQLLIRKHRNSS alpha actinin-2 2773059 1QTVILPGPAAWGFRLSGGIDFNQPLVITRITPGSKAAAANLCPGDVILAIDGFGTESMTHADGQ 7associated DRIKAAEFIV LIM protein APXL-1 1365126 1ILVEVQLSGGAPWGFTLKGGREHGEPLVITKIEEGSKAAAVDKLLAGDEIVGINDIGLSGFRQ 8EAICLVKGSHKTLKLVVKRNSS Atrophin-1 2947231 1REKPLFTRDASQLKGTFLSTTLKKSNMGFGFTIIGGDEPDEFLQVKSVIPDGPAAQDGKMETG 9Interacting DVIVVINEVCVLGHTHADVVKLFQSVPIGQSVNLVLCRGYP Protein Atrophin-12947231 2LSGATQAELMTLTIVKGAQGFGFTIADSPTGQRVKQILDIQGCPGLCEGDLIVEINQQNVQNL 10Interacting SHTEVVDILKDCPIGSETSLIIHRGGFF Protein Atrophin-1 2947231 3HYKELDVHLRRMESGFGFRILGGDEPGQPILIGAVIAMGSADRDGRLHPGDELVYVDGIPVAG 11Interacting KTHRYVIDLMHHAARNGQVNLTVRRKVLCG Protein Atrophin-1 2947231 4EGRGISSHSLQTSDAVIHRKENEGFGFVIISSLNRPESGSTITVPHKIGRIIDGSPADRCAKL 12Interacting KVGDRILAVNGQSIINMPHADIVKLIKDAGLSVTLRIIPQEEL ProteinAtrophin-1 2947231 5LSDYRQPQDFDYFTVDMEKGAKGFGFSIRGGREYKMDLYVLRLAEDGPAIRNGRMRVGDQIIE 13Interacting INGESTRDMTHARAIELIKSGGRRVRLLLKRGTGQ Protein Atrophin-12947231 6HESVIGRNPEGQLGFELKGGAENGQFPYLGEVKPGKVAYESGSKLVSEELLLEVNETPVAGLT 14Interacting IRDVLAVIKHCKDPLRLKCVKQGGIHR Protein CARD11 12382772 1NLMFRKFSLERPFRPSVTSVGHVRGPGPSVQHTTLNGDSLTSQLTLLGGNARGSFVHSVKPGS 15KLAEKAGLREGHQLLLLEGCIRGERQSVPLDTCTKEEAHWTIQRCSGPVTLHYKVNHEGYRKL V CARD1413129123 1ILSQVTMLAFQGDALLEQISVIGGNLTGIFIHRVTPGSAADQMALRPGTQIVMVDYEASEPLF 16KAVLEDTTLEEAVGLLRRVDGFCCLSVKVNTDGYKRL CASK 3087815 1TRVRLVQFQKNTDEPMGITLKMNELNHCIVARIMHGGMIHRQGTLHVGDEIREINGISVANQTV 17EVQLQKMLREMRGSITPKIVPSYRTQS Connector 3930780 1LEQKAVLEQVQLDSPLGLEIHTTSNCQHFVSQVDTQVPTDSRLQIQPGDEVVQINEQVVVGWPR 18Enhancer KNMVRELLREPAGLSLVLKKIPIP Cytohesin 3192908 1QRKLVTVEKQDNETFGFEIQSYRPQNQNACSSEMFTLICKIQEDSPAHCAGLQAGDVLANING 19Binding VSTEGFTYKQVVDLIRSSGNLLTIETLNG Protein Densin 180 16755892 1RCLIQTKGQRSMDGYPEQFCVRIEKNPGLGFSISGGISGQGNPFKPSDKGIFVTRVQPDGPAS 20NLLQPGDKILQANGHSFVHMEHEKAVLLLKSFQNTVDLVIQRELTV DLG1 475816 1IQVNGTDADYEYEEITLERGNSGLGFSIAGGTDNPHIGDDSSIFITKIITGGAAAQDGRLRVN 21DCILQVNEVDVRDVTHSKAVEALKEAGSIVRLYVKRRN DLG1 475816 2IQLIKGPKGLGFSIAGGVGNQHIPGDNSIYVTKIIEGGAAHKDGKLQIGDKLLAVNNVCLEEV 22THEEAVTALKNTSDFVYLKVAKPTSMYMNDGN DLG1 475816 3ILHRGSTGLGFNIVGGEDGEGIFISFILAGGPADLSGELRKGDRIISVNSVDLRAASHEQAAA 23ALKNAGQAVTIVAQYRPEEYSR DLG2 12736552 1ISYVNGTEIEYEFEEITLERGNSGLGFSIAGGTDNPHIGDDPGIFITKIIPGGAAAEDGRLRV 24NDCILRVNEVDVSEVSHSKAVEALKEAGSIVRLYVRRR DLG2 12736552 2ISVVEIKLFKGPKGLGFSIAGGVGNQHIPGDNSIYVTKIIDGGAAQKDGRLQVGDRLLMVNNYSL 25EEVTHEEAVAILKNTSEVVYLKVGNPTTI DLG2 12736552 3IWAVSLEGEPRKVVLHKGSTGLGFNIVGGEDGEGIFVSFILAGGPADLSGELQRGDQILSVNGI 26DLRGASHEQAAAALKGAGQTVTIIAQYQPED DLG5 3650451 1GIPYVEEPRHVKVQKGSEPLGISIVSGEKGGIYVSKVTVGSIAHQAGLEYGDQLLEFNGINLR 27SATEQQARLIIGQQCDTITILAQYNPHVHQLRNSSZLTD DLG5 3650451 2GILAGDANKKTLEPRVVFIKKSQLELGVHLCGGNLHGVFVAEVEDDSPAKGPDGLVPGDLILE 28YGSLDVRNKTVEEVYVEMLKPRDGVRLKVQYRPEEFIVTD DLG6, splice 14647140 1PTSPEIQELRQMLQAPHFKALLSAHDTIAQKDFEPLLPPLPDNIPESEEAMRIVCLVKNQQPL 29variant 1GATIKRHEMTGDILVARIIHGGLAERSGLLYAGDKLVEVNGVSVEGLDPEQVIHILAMSRGTIMFKVVPVSDPPVNSS DLG6 splice AB053303 1PTSPEIQELRQMLQAPHFKGATIKRHEMTGDILVARIIHGGLAERSGLLYAGDKLVEVNGVSV 30variant 2 EGLDPEQVIHILAMSRGTIMFKVVPVSDPPVNSS DVL1 2291005 1LNIVTVTLNMERHHFLGISIVGQSNDRGDGGIYIGSIMKGGAVAADGRIEPGDMLLQVNDVNF 31ENMSNDDAVRVLREIVSQTGPISLTVAKCW DVL2 2291007 1LNIITVTLNMEKYNFLGISIVGQSNERGDGGIYIGSIMKGGAVAADGRIEPGDMLLQVNDMNF 32ENMSNDDAVRVLRDIVHKPGPIVLTVAKCWDPSPQNS DVL3 6806886 1IITVTLNMEKYNFLGISIVGQSNERGDGGIYIGSIMKGGAVAADGRIEPGDMLLQVNEINFEN 33MSNDDAVRVLREIVHKPGPITLTVAKCWDPSP ELFIN 1 2957144 1TTQQIDLQGPGPWGFRLVGRKDFEQPLAISRVTPGSKAALANLCIGDVITAIDGENTSNMTHL 34EAQNRIKGCTDNLTVARSEHKVWSPLV ENIGMA 561636 1IFMDSFKVVLEGPAPWGFRLQGGKDFNVPLSISRLTPGGKAAQAGVAVGDWVLSIDGENAGSL 35THIEAQNKIRACGERLSLGLSRAQPV ERBIN 8923908 1QGHELAKQEIRVRVEKDPELGFSISGGVGGRGNPFRPDDDGIFVTRVQPEGPASKLLQPGDKI 36IQANGYSFINIEHGQAVSLLKTFQNTVELIIVREVSS EZRIN Binding 3220018 1ILCCLEKGPNGYGFHLHGEKGKLGQYIRLVEPGSPAEKAGLLAGDRLVEVNGENVEKETHQQV 37Protein 50 VSRIRAALNAVRLLVVDPEFIVTD EZRIN Binding 3220018 2IRLCTMKKGPSGYGFNLHSDKSKPGQFIRSVDPDSPAEASGLRAQDRIVEVNGVCMEGKQHGD 38Protein 50 VVSAIRAGGDETKLLVVDRETDEFFMNSS FLJ00011 10440352 1KNPSGELKTVTLSKMKQSLGISISGGIESKVQPMVKIEKIFPGGAAFLSGALQAGFELVAVDG 39ENLEQVTHQRAVDTIRRAYRNKAREPMELVVRVPGPSPRPSPSD FLJ11215 11436365 1EGHSHPRVVELPKTEEGLGFNIMGGKEQNSPIYISRIIPGGIADRHGGLKRGDQLLSVNGVSV 40EGEHHEKAVELLKAAQGKVKLVVRYTPKVLEEME FLJ12428 BC012040 1PGAPYARKTFTIVGDAVGWGFVVRGSKPCHIQAVDPSGPAAAAGMKVCQFVVSVNGLNVLHVD 41YRTVSNLILTGPRTIVMEVMEELEC FLJ12615 10434209 1GQYGGETVKIVRIEKARDIPLGATVRNEMDSVIISRIVKGGAAEKSGLLHEGDEVLEINGIEI 42RGKDVNEVFDLLSDMHGTLTFVLIPSQQIKPPPA FLJ20075 7019938 1ILAHVKGIEKEVNVYKSEDSLGLTITDNGVGYAFIKRIKDGGVIDSVKTICVGDHIESINGEN 43IVGWRHYDVAKKLKELKKEELFTMKLIEPKKAFEI FLJ21687 10437836 1KPSQASGHFSVELVRGYAGFGLTLGGGRDVAGDTPLAVRGLLKDGPAQRCGRLEVGDLVLHIN 44GESTQGLTHAQAVERIRAGGPQLHLVIRRPLETHPGKPRGV FLJ31349 AK055911 1PVMSQCACLEEVHLPNIKPGEGLGMYIKSTYDGLHVITGTTENSPADRSQKIHAGDEIVQVNQ 45QVVGWQLKNLVKKLRENPTGVVLLLKKRPTGSFNFTPEFIVTD FLJ32798 AK057360 1LDDEEDSVKIIRLVKNREPLGATIKKDEQTGAIIVARIMRGGAADRSGLIHVGDELREVNGIP 45VEDKRPEEIIQILAQSQGAITFKIIPGSKEETPSNSS GRIP 1 4539083 1VVELMKKEGTTLGLTVSGGIDKDGKPRVSNLRQGGIAARSDQLDVGDYIKAVNGINLAKFRH 47DEIISLLKNVGERVVLEVEYE GRIP 1 4539083 2RSSVIFRTVEVTLHKEGNTFGFVIRGGAHDDRNKSRPVVITCVRPGGPADREGTIKPGDRLLS 48VDGIRLLGTTHAEAMSILKQCGQEAALLIEYDVSVMDSVATASGNSS GRIP 1 4539083 3HVATASGPLLVEVAKTPGASLGVALTTSMCCNKQVIVIDKIKSASIADRCGALHVGDHILSID 49GTSMEYCTLAEATQFLANTTDQVKLEILPHHQTRLALKGPNSS GRIP 1 4539083 4TETTEVVLTADPVTGFGIQLQGSVFATETLSSPPLISYIEADSPAERCGVLQIGDRVMAINGI 50PTEDSTFEEASQLLRDSSITSKVTLEIEFDVAES GRIP 1 4539083 5AESVIPSSGTFHVKLPKKHNVELGITISSPSSRKPGDPLVISDIKKGSVAHRTGTLELGDKLL 51AIDNIRLDNCSMEDAVQILQQCEDLVKLKIRKDEDNSD GRIP 1 4539083 6IYTVELKRYGGPLGITISGTEEPFDPIIISSLTKGGLAERTGAIHIGDRILAINSSSLKGKPL 52SEAIHLLQMAGETVTLKIKKQTDAQSA GRIP 1 4539083 7IMSPTPVELHKVTLYKDSDMEDFGFSVADGLLEKGVYVKNIRPAGPGDLGGLKPYDRLLQVNH 53VRTRDFDCCLVVPLIAESGNKLDLVISRNPLA GTPase 2389008 1SRGCETRELALPRDGQGRLGFEVDAEGFVTHVERFTFAETAGLRPGARLLRVCGQTLPSLRPE 54Activating AAAQLLRSAPKVCVTVLPPDESGRP Enzyme Guanine 6650765 1AKAKVVRQWLQKASRESPLQFSLNGGSEKGFGIFVEGVEPGSKAADSGLKRGDQIMEVNGQNF 55Exchange ENITFMKAVEILRNNTHLALTVKTNIFVFKEL Factor HEMBA 10436367 1LENVIAKSLLIKSNEGSYGFGLEDKNKVPIIKLVEKGSNAEMAGMEVGKKIFAINGDLVFMRP 561000505 FNEVDCFLKSCLNSRKPLRVLVSTKP HEMBA 10436367 2PRETVKIPDSADGLGFQIRGFGPSVVHAVGRGTVAAAAGLHPGQCIIKVNGINVSKETHASVI 571000505 AHVTACRKYRRPTKQDSIQ HEMBA 7022001 1EDFCYVFTVELERGPSGLGMGLIDGMHTHLGAPGLYIQTLLPGSPAAADGRLSLGDRILEVNG 581003117 SSLLGLGYLRAVDLIRHGGKKMRFLVAKSDVETAKKI HTRA3 AY040094 1LTEFQDKQIKDWKKRFIGIRMRTITPSLVDELKASNPDFPEVSSGIYVQEVAPNSPSQRGGIQ 59DGDIIVKVNGRPLVDSSELQEAVLTESPLLLEVRRGNDDLLFSNSS HTRA4 AL576444 1HKKYLGLQMLSLTVPLSEELKMHYPDFPDVSSGVVVCKVVEGTAAQSSGLRDHDVIVNINGKP 60ITTTTDVVKALDSDSLSMAVLRGKDNLLLTVNSS INADL 2370148 1IWQIEYIDIERPSTGGLGFSVVALRSQNLGKVDIFVKDVQPGSVADRDQRLKENDQILAINHT 61PLDQNISHQQAIALLQQTTGSLRLIVAREPVHTKSSTSSSE INADL 2370148 2PGHVEEVELINDGSGLGFGIVGGKTSGVVVRTIVPGGLADRDGRLQTGDHILKIGGTNVQGMT 62SEQVAQVLRNCGNSS INADL 2370148 3PGSDSSLFETYNVELVRKDGQSLGIRIVGYVGTSHTGEASGIYVKSIIPGSAAYHNGHIQVND 63KIVAVDGVNIQGFANHDVVEVLRNAGQVVHLTLVRRKTSSSTSRIHRD INADL 2370148 4NSDDAELQKYSKLLPIHTLRLGVEVDSFDGHHYISSIVSGGPVDTLGLLQPEDELLEVNGMQL 64YGKSRREAVSFLKEVPPPFTLVCCRRLFDDEAS INADL 2370148 5LSSPEVKIVELVKDCKGLGFSILDYQDPLDPTRSVIVIRSLVADGVAERSGGLLPGDRLVSVN 65EYCLDNTSLAEAVEILKAVPPGLVHLGICKPLVEFIVTD INADL 2370148 6PNFSHWGPPRIVEIFREPNVSLGISIVVGQTVIKRLKNGEELKGIFIKQVLEDSPAGKTNALK 66TGDKILEVSGVDLQNASHSEAVEAIKNAGNPVVFIVQSLSSTPRVIPNVHNKANSS INADL 2370148 7PGELHIIELEKDKNGLGLSLAGNKDRSRMSIFVVGINPEGPAAADGRMRIGDELLEINNQILY 67GRSHQNASAIIKTAPSKVKLVFIRNEDAVNQMANSS INADL 2370148 8PATCPIVPGQEMIIEISKGRSGLGLSIVGGKDTPLNAIVIHEVYEEGAAARDGRLWAGDQILE 68VNGVDLRNSSHEEAITALRQTPQKVRLVVY KIAA0147 1469875 1ILTLTILRQTGGLGISIAGGKGSTPYKGDDEGIFISRVSEEGPAARAGVRVGDKLLEVNGVAL 69QGAEHHEAVEALRGAGTAVQMRVWRERMVEPENAEFIVTD KIAA0147 1469875 2PLRQRHVACLARSERGLGFSIAGGKGSTPYRAGDAGIFVSRIAEGGAAHRAGTLQVGDRVLSI 70NGVDVTEARHDHAVSLLTAASPTIALLLEREAGG KIAA0147 1469875 3ILEGPYPVEEIRLPRAGGPLGLSIVGGSDHSSHPFGVQEPGVFISKVLPRGLAARSGLRVGDR 71ILAVNGQDVRDATHOEAVSALLRPCLELSLLVRRDPAEFIVTD KIAA0147 1469875 4RELCIQKAPGERLGISIRGGARGHAGNPRDPTDEGIFISKVSPTGAAGRDGRLRVGLRLLEVN 72QQSLLGLTHGEAVQLLRSVGDTLTVLVCDGFEASTDAALEVS KIAA0303 2224546 1PHQPIVIHSSGKNYGFTIRAIRVYVGDSDIYTVHHIVWNVEEGSPACQAGLKAGDLITHINGE 73PVHGLVHTEVIELLLKSGNKVSITTTPF KIAA0313 7657260 1ILACAAKAKRRLMTLTKPSREAPLPFILLGGSEKGFGIFVDSVDSGSKATEAGLKRGDQILEV 74NGQNFENIQLSKAMEILRNNTHLSITVKTNLFVFKELLTNSS KIAA0316 6683123 1IPPAPRKVEMRRDPVLGFGFVAGSEKPVVVRSVTPGGPSEGKLIPGDQIVMINDEPVSAAPRE 75RVIDLVRSCKESILLTVIQPYPSPK KIAA0340 2224620 1LNKRTTMPKDSGALLGLKVVGGKMTDLGRLGAFITKVKKGSLADVVGHLRAGDEVLEWNGKPL 76PGATNEEVYNIILESKSEPQVEIIVSRPIGDIPRIHRD KIAA0380 2224700 1QRCVIIQKDQHGFGFTVSGDRIVLVQSVRPGGAAMKAGVKEGDRIIKVNGTMVTNSSHLEVVK 77LIKSGAYVALTLLGSS KIAA0382 7662087 1ILVQRCVIIQKDDNGFGLTVSGDNPVFVQSVKEDGAAMRAGVQTGDRIIKVNGTLVTHSNHLE 78VVKLIKSGSYVALTVQGRPPGNSS KIAA0440 2662160 1SVEMTLRRNGLGQLGFHVNYEGIVADVEPYGYAWQAGLRQGSRLVEICKVAVATLSHEQMIDL 79LRTSVTVKVVIIPPHD KIAA0545 14762850 1LKVMTSGWETVDMTLRRNGLGQLGFHVKYDGTVAEVEDYGFAWQAGLRQGSRLVEICKVAVVT 80LTHDQMIDLLRTSVTVKVVIIPPFEDGTPRRGW KIAA0559 3043641 1HYIFPHARIKITRDSKDHTVSGNGLGIRIVGGKEIPGHSGEIGAYIAKILPGGSAEQTGKLME 81GMQVLEWNGIPLTSKTYEEVQSIISQQSGEAEICVRLDLNML KIAA0561 3043645 1LCGSLRPPIVIHSSGKKYGFSLRAIRVYMGDSDVYTVHHVVWSVEDGSPAQEAGLRAGDLITH 82INGESVLGLVHMDVVELLLKSGNKISLRTTALENTSIKVG KIAA0613 3327039 1SYSVTLTGPGPWGFRLQGGKDFNMPLTISRITPGSKAAQSQLSQGDLVVAIDGVNTDTMTHLE 83AQNKIKSASYNLSLTLQKSKNSS KIAA0751 12734165 1ISRDSGAMLGLKVVGGKMTESGRLCAFITKVKKGSLADTVGHLRPGDEVLEWNGRLLQGATFE 84EVYNIILESKPEPQVELVVSRPIAIHRD KIAA0807 3882334 1ISALGSMRPPIIIHRAGKKYGFTLRAIRVYMGDSDVYTVHHMVWHVEDGGPASEAGLRQGDLI 85THVNGEPVHGLVHTEVVELILKSGNKVAISTTPLENSS KIAA0858 4240204 1FSDMRISINQTPGKSLDFGFTIKWDIPGIFVASVEAGSPAEFSQLQVDDEIIAINNTKFSYND 86SKEWEEAMAKAQETGHLVMDVRRYGKAGSPE KIAA0902 4240292 1QSAHLEVIQLANIKPSEGLGMYIKSTYDGLHVITGTTENSPADRCKKIHAGDEVIQVNHQTVV 87GWQLKNLVNALREDPSGVILTLKKRPQSMLTSAPA KIAA0967 4589577 1ILTQTLIPVRHTVKIDKDTLLQDYGFHISESLPLTVVAVTAGGSAHGKLFPGDQILQMNNEPA 88EDLSWERAVDILREAEDSLSITVVRCTSGVPKSSNSS KIAA0973 4589589 1GLRSPITIQRSGKKYGFTLRAIRVYMGDTDVYSVHHIVWHVEEGGPAQEAGLCAGDLITHVNG 89EPVHGMVHPEVVELILKSGNKVAVTTTPFE KIAA1095 5889526 1QGEETKSLTLVLHRDSGSLGFNIIGGRPSVDNHDGSSSEGIFVSKIVDSGPAAKEGGLQIHDR 90IIEVNGRDLSRATHDQAVEAFKTAKEPIVVQVLRRTPRTKMP KIAA1095 5889526 2QEMDREELELEEVDLYRMNSQDKLGLTVCYRTDDEDDIGIYISEIDPNSIAAKDGRIREGDRI 91IQINGIEVQNREEAVALLTSEENKNFSLLIARPELQLD KIAA1202 6330421 1RSFQYVPVQLQGGAPWGFTLKGGLEHCEPLTVSKIEDGGKAALSQKMRTGDELVNINGTPLYG 92SRQEALILIKGSFRILKLIVRRRNAPVS KIAA1222 6330610 1ILEKLELFPVELEKDEDGLGISIIGMGVGADAGLEKLGIFVKTVTEGGAAQRDGRIQVNDQIV 93EVDGISLVGVTQNFAATVLRNTKGNVRFVIGREKPGQVS KIAA1284 6331369 1KDVNVYVNPKKLTVIKAKEQLKLLEVLVGIIHQTKWSWRRTGKQGDGERLWHGLLPGGSAMKS 94GQVLIGDVLVAVNDVDVTTENIERVLSCIPGPMQVKLTFENAYDVKRET KIAA1389 7243158 1TRGCETVEMTLRRNGLGQLGFHVNFEGIVADVEPFGFAWKAGLRQGSRLVEICKVAATLTHEQ 95MIDLLRTSVTVKVVIIQPHDDGSPRR KIAA1415 7243210 1VENILAKRLLILPQEEDYGFDIEEKNKAVVVKSVQRGSLAEVAGLQVGRKIYSINEDLVFLRP 96FSEVESILNQSFCSRRPLRLLVATKAKEIIKIP KIAA1526 5817166 1PDSAGPGEVRLVSLRRAKAHEGLGFSIRGGSEHGVGIYVSLVEPGSLAEKEGLRVGDQILRVN 97DKSLARVTHAEAVKALKGSKKLVLSVYSAGRIPGGYVTNH KIAA1526 5817166 2LQGGDEKKVNLVLGDGRSLGLTIRGGAEYGLGIYITGVDPGSEAEGSGLKVGDQILEVNWRSF 98LNILHDEAVRLLKSSRHLILTVKDVGRLPHARTTDV KIAA1526 5817166 3WTSGAHVHSGPCEEKCGHPGHRQPLPRIVTIQRGGSAHNCGQLKVGHVILEVNGLTLRGKEHR 99EAARIIAEAFKTKDRDYIDFLDSL KIAA1620 10047316 1ELRRAELVEIIVETEAQTGVSGINVAGGGKEGIFVRELREDSPAARSLSLQEGDQLLSARVFF 100ENFKYEDALRLLQCAEPYKVSFCLKRTVPTGDLALRP KIAA1634 10047344 1PSQLKGVLVRASLKKSTMGFGFTIIGGDRPDEFLQVKNVLKDGPAAQDGKIAPGDVIVDINGN 101CVLGHTHADVVQMFQLVPVNQYVNLTLCRGYPLPDDSED KIAA1634 10047344 2ASSGSSQPELVTIPLIKGPKGFGFAIADSPTGQKVKMILDSQWCQGLQKGDIIKEIYHQNVQN 102LTHLQVVEVLKQFPVGADVPLLILRGGPPSPTKTAKM KIAA1634 10047344 3LYEDKPPLTNTFLISNPRTTADPRILYEDKPPNTKDLDVFLRKQESGFGFRVLGGDGPDQSIY 103IGAIIPLGAAEKDGRLRAADELMCIDGIPVKGKSHKQVLDLMTTAARNGHVLLTVRRKIFYGEKQPEDDSGSPGIHRELT KIAA1634 10047344 4PAPQEPYDVVLQRKENEGFGFVILTSKNKPPPGVIPHKIGRVIEGSPADRCGKLKVGDHISAV 104NGQSIVELSHDNIVQLIKDAGVTVTLTVIAEEEHHGPPS KIAA1634 10047344 5QNLGCYPVELERGPRGFGFSLRGGKEYNMGLFILRLAEDGPAIKDGRIHVGDQIVEINGEPTQ 105GITHTRAIELIQAGGNKVLLLLRPGTGLIPDHGLA KIAA1719 1267982 0ITVVELIKKEGSTLGLTISGGTDKDGKPRVSNLRPGGLRSDLLNIGDYIRSVNGIHLTRLRHD 106EIITLLKNVGERVVLEVEY KIAA1719 1267982 1ILDVSLYKEGNSFGFVLRGGAHEDGHKSRPLVLTYVRPGGPADREGSLKVGDRLLSVDGIPLH 107GASHATALATLRQCSHEALFQVEYDVATP KIAA1719 1267982 2IHTVANASGPLMVEIVKTPGSALGISLTTTSLRNKSVITIDRIKPASVVDRSGALHPGDHIL 108SIDTSMEHCSLLEATKLLASISEKVRLEILPVPQSQRPL KIAA1719 1267982 3IQIVHTETTEVVLCGDPLSGFGLQLQGGIFATETLSSPPLVCFIEPDSPAERCGLLQVGDRVL 109SINGIATEDGTMEEANQLLRDAALAHKVVLEVEFDVAESV KIAA1719 1267982 4IQFDVAESVIPSSGTFHVKLPKKRSVELGITISSASRKRGEPLIISDIKKGSVAHRTGTLEPG 110DKLLAIDNIRLDNCPMEDAVQILRQCEDLVKLKIRKKDEDN KIAA1719 1267982 5IQTTGAVSYTVELKRYGGPLGITISGTEEPFDPIVISGLTKRGLAERTGAIHVGDRILAINNV 111SLKGRPLSEAIHLLQVAGETVTLKIKKQLDR KIAA1719 1267982 6ILEMEELLLPTPLEMHKVTLHKDPMRHDPGFSVSDGLLEKGVYVHTVRPDGPAHRGGLQPFDR 112VLQVNHVRTRDPDCCLAVPLLAEAGDVLELIISRKPHTAHSS LIM Mystique 12734250 1MALTVDVAGPAPWGFRITGGRDFHTPIMVTKVAERGKAKDADLRPGDIIVAINGESAQGMLHA 113EAQSKIRQSPSPLRLQLDRSQATSPGQT LIM Protein 3108098 1TNYSVSLVGPAPWGFRLQGGKDFNMPLTISSLKDGGKAAQANVRIGDWLSIDGINAGMTHLEA 114QNKIKGCTGSLNMTLQRAS LIMK1 4587498 1TLVEHSKLYCGHCYYQTVVTPVIEQILPDSPGSHLPHTVTLVSIPASSHGKRGLSVSIDPPHG 115PPGCGTEHSHTVRVQGVDPGCMSPDVKNSIHVGORILEINGTPIRNVPLDEIDLLIQETSRLL QLTLEHDLIMK2 1805593 1PYSVTLISMPATTEGRRGFSVSVESACSNYATTVQVKEVNRMHISPNNRNAIHPGDRILEING 116TPVRTLRVEEVEDAISQTSQTLQLLIEHD LIM-RIL 1085021 1IHSVTLRGPSPWGFRLVGRDFSAPLTISRVHAGSKASLAALCPGDLIQAINGESTELMTHLEA 117QNRIKGCHDHLTLSVSRPE LU-1 U52111 1VCYRTDDEEDLGIYVGEVNPNSIAAKDGRIREGDRIIQINGVDVQNREEAVAILSQEENTNIS 118LLVARPESQLA MAGI1 3370997 1IQKKNHWTSRVHECTVKRGPQGELGVTVLGGAEHGEFPYVGAVAAVEAAGLPGGGEGPRLGEG 119ELLLEVQGVRVSGLPRYDVLGVIDSCKEAVTFKAVRQGGR MAGI1 3370997 2PSELKGKFIHTKLRKSSRGFGFTVVGGDEPDEFLQIKSLVLDGPAALDGKMETGDVIVSVNDT 120CLGHTHAQVVKIFQSIPIGASVDLELCRGYPLPFDPDDPN MAGI1 3370997 3PATQPELITVHIVKGPMGFGFTIADSPGGGGQRVKQIVDSPRCRGLKEGDLIVEVNKKNVQAL 121THNQVVDMLVECPKGSEVTLLVQRGGNLS MAGI1 3370997 4PDYQEQDIFLWRKETGFGFRILGGNEPGEPIYIGHIVPLGAADTDGRLRSGDELICVDGTPVI 122GKSHQLWQLMQQAAKQGHVNLTVRRKVVFAVPKTENSS MAGI1 3370997 5GVVSTVVQPYDVEIRRGENEGFGFVIVSSVSRPEAGTTFAGNACVAMPHKIGRIIEGSPADRC 123GKLKVGDRILAVNGCSITNKSHSDIVNLIKEAGNTVTLRIIPGDESSNA MAGI1 3370997 6QATQEQDFYTVELERGAKGFGFSLRGGREYNMDLYVLRLAEDGPAERCGKMRIGDEILEINGE 124TTKNMKHSRAIELIKNGGRRVRLFLKRG MGC5395 BC012477 1PAKMEKEETTRELLLPNWQGSGSHGLTIAQRDDGVFVQEVTQNSPAARTGVVKEGDQIVGATI 125YFDNLQSGEVTQLLNTMGHHTVGLKLHRKGDRSPNSS MINT1 2625024 1SENCKdVFIEKQKGEILGVVIVESGWGSILPTVIIANMMHGGPAEKSGKLNIGDQIMSINGTS 126LVGLPLSTCQSIIKGLKNQSRVKLNIVRCPPVNSS MINT1 2625024 2LRCPPVTTVLIRRPDLRYQLGSVQNGIICSLMRGGIAERGGVRVGHRIIEINGQSVVATPHEK 127IVHILSNAVGEIHMKTMPAAMYRLLNSS MINT3 3169808 1LSNSDNCREVHLEKRRGEGLGVALVESGWGSLLPTAVIANLLHGGPAERSGALSIGDRLTAIN 128GTSLVGLPLAACQAAVRETKSQTSVTLSIVHCPPVTTAIM MINT3 3169808 2LVHCPPVTTAIIHRPHAREQLGFCVEDGIICSLLRGGIAERGGIRVGHRIIEINGQSVVATPH 129ARIIELLTEAYGEVHIKTMPAATYRLLTG MPP1 189785 1RKVRLIQFEKVTEEPMGITLKLNEKQSCTVARILHGGMIHRQGSLHVGDEILEINGTNVTNHS 130VDQLQKAMKETKGMISLKVIPNQ MPP2 939884 1PVPPDAVRMVGIRKTAGEHLGVTFRVEGGELVIARILHGGMVAQQGLLHVGDIIKEVNGQPVG 131SDPRALQELLRNASGSVILKILPNYQ MUPP1 2104784 1GRHVEVFELLKPPSGGLGFSVVGLRSENRGELGIFVQEIQEGSVAHRDGRLKETDQILAINGQ 132ALDQTITHQQAISILQKAKDTVQLVIARGSLPQLV MUPP1 2104784 2PVHWQHMETIELVNDGSGLGFGIIGGKATGVIVKTILPGGVADQHGRLCSGDHILKIGDTDLA 133GMSSEQVAQVLRQCGNRVKLMIARGAIEERTAPT MUPP1 2104784 3QESETPDVELTKNVQGLGITIAGYIGDKKLEPSGIFVKSITKSSAVEHDGRIQIGDQIIAVDG 134TNLQGFTNQQAVEVLRHTGQTVLLTLMRRGMKQEA MUPP1 2104784 4LNYEIVVAHVSKFSENSGLGISLEATVGHHFIRSVLPEGPVGHSGKLFSGDELLEVNGITLL 135GENHQDVVNILKELPIEVTMVCCRRTVPPT MUPP1 2104784 5WEAGIQHIELEKGSKGLGFSILDYQDPIDPASTVIIIRSLVPGGIAEKDGRLLPGDRLMFVNDV 136NLENSSLEEAVEALKGAPSGTVRIGVAKPLPLSPEE MUPP1 2104784 6RNVSKESFERTINIAKGNSSLGMTVSANKDGLGMIVRSIIHGGAISRDGRIAIGDCILSINEE 137STISVTNAQARAMLRRHSLIGPDIKITYVPAEHLEE MUPP1 2104784 7LNWNQPRRVELWREPSKSLGISIVGGRGMGSRLSNGEVMRGIFIKHVLEDSPAGKNGTLKPGD 138RIVEVDGMDLRDASHEQAVEAIRKAGNPVVFMVQSIINRPRKSPLPSLL MUPP1 2104784 8LTGELHMIELEKGHSGLGLSLAGNKDRSRMSVFIVGIDPNGAAGKDGRLQIADELLEINGQIL 139YGRSHQNASSIIKCAPSKVKIIFIRNKDAVNQ MUPP1 2104784 9LSSFKNVQHLELPKDQGGLGIAISEEDTLSGVIIKSLTEHGVAATDGRLKVGDQILAVDDEIV 140VGYPIEKFISLLKTAKMTVKLTIHAENPDSQ MUPP1 2104784 10LPGCETTIEISKGRTGLGLSIVGGSDTLLGAIIIHEVYEEGAACKDGRLWAGDQILEVNGIDL 141RKATHDEAINVLRQTPQRVRLTLYRDEAPYKE MUPP1 2104784 11KEEEVCDTLTIELQKKPGKGLGLSIVGKRNDTGVFVSOIVKGGIADADGRLMQGDQILMVNGE 142DVRNATQEAVAALLKCSLGTVTLEVGRIKAGPFHS MUPP1 2104784 12LQGLRTVEMKKGPTDSLGISAGGVGSPLGDVPIFIAMMHPTGVAAQTQKLRVGDRIVTICGTS 143TEGMTHTQAVNLLKNASGSIEMQVVAGGDVSV MUPP1 2104784 13LGPPQCKSITLERGPDGLGFSIVGGYGSPHGDLPIYVKTVFAKGAASEDGRLKRGDQIIAVNG 144QSLEGVTHEEAVAILKRTKGTVTLMVLS NeDLG 10863920 1IQYEEIVLERGNSGLGFSIAGGIDNPHVPDDPGIFITKIIPGGAAAMDGRLGVNDCVLRVNEV 145EVSEVVHSRAVEALKEAGPVVRLVVRRRQN NeDLG 10863920 2ITLLKGPKGLGFSIAGGIGNQHIPGONSIYITKIIEGGAAQKDGRLQIGDRLLAVNNTNLQDV 146RHEEAVASLKNTSDMVYLKVAKPGSLE NeDLG 10863920 3ILLHKGSTGLGFNIVGGEDGEGIFVSFILAGGPADLSGELRRGDRILSVNGVNLRNAT 147HEQAAAALKRAGQSVTIVAQYRPEEYSRFESKIHDLREQMMNSSMSSGSGSLRTSE KRSLE NeurabinII AJ401189 1 CVERLELFPVELEKDSEGLGISIGMGAGADMGLEKLGIFVKTVTEGGAAHRDGRIQV148 NDLLVEVDGTSLVGVTQSFAASVLRNTKGRVRFMIGRERPGEQSEVAQRIHRD NOS1 642424 1IQPNVISVRLFKRKVGGLGFLVKERVSKPPVIISDLIRGGAAEQSGLIQAGDIILAVNGR 149PLVDLSYDSALEVLRGIASETHVVLILRGP novel PDZ 7228177 1QANSDESDIIHSVRVEKSPAGRLGFSVRGGSEHGLGIFVSKVEEGSSAERAGLCVGDKITEVN 150 geneGLSLESTTMGSAVKVLTSSSRLHMMVRRMGRVPGIKFSKEKNSS novel PDZ 7228177 2PSDTSSEDGVRRIVHLYTTSDDFCLGFNIRGGKEFGLGIYVSKVDHGGLAEENGIKVGDQVLA 151 geneANGVRFDDISHSQAVEVLKGQTHIMLTIKETGRYPAYKEMNSS Novel Serin 1621243 1LIKKFLTESHDRQAKGKAITKKKYIGIRMMSLTSSKAKELKDRHRDFPDVISGAYIIEVIPDT 152Protease PAEAGGLKENDVIISINGQSVVSANDVSDVIKRESTLNMVVRRGNEDIMITV NumbBinding AK056823 1PDGEITSIKINRVDPSESLSIRLVGGSETPLVHIIIQHIYRDGVIARDGRLLPGDIILKVNGM 153Protein DISNVPHNYAVRLLRQPCQVLWLTVMREQKFRSRNSS Numb Binding AK056823 2HRPRDDSFHVILNKSSPEEQLGIKLVRKVDEPGVFIFNVLDGGVAYRHGQLEENDRVLAINGH 154Protein DLRYGSPESAAHLIQASERRVHLVVSRQVRQRSPENSS Numb Binding AK056823 3PTITCHEKVVNIQKDPGESLGMTVAGGASHREWDLPIYVISVEPGGVISRDGRIKTGDILLN 155Protein VDGVELTEVSRSEAVALLKRTSSSIVLKALEVKEYEPQEFIV Numb Binding AK0568234 PRCLYNCKDIVLRRNTAGSLGFCIVGGYEEYNGNKPPFIKSIVEGTPAYNDGRIRCGDILLAV 156Protein NGRSTSGMIHACLARLLKELKGRITIVSWPGTPL Outer 7023825 1LLTEEEINLTRGPSGLGFNIVGGTDQQYVSNDSGIYVSRIKENGAAALDGRLQEGDKILSVNG 157Membrane QDLKNLLHQDAVDLFRNAGYAVSLRVQHRLQVQNGIHS p55T 12733367 1PVDAIRILGIHKRAGEPLGVTFRVENNDLVIARILHGGMIDRQGLLHVGDIIKEVNGHEVGNN 158PKELQELLKNISGSVTLKILPSYRDTITPQQ PAR3 8037914 1DDMVKLVEVPNDGGPLGIHVVPFSARGGRTLGLLVKRLEKGGKAEHENLFRENDCIVRINDGD 159LRNRRPEQAQHMFRQAMRTPIIVVPHWPAA PAR3 8037914 2GKRLNIQLKKGTEGLGFSITSRDVTIGGSAPIYVKNILPRGAAIQDGRLKAGDRLIEVNGVDL 160VGKSQEEVVSLLRSTKMEGTVSLLVFRQEDA PAR3 8037914 3TPDGTREPLTFEVPLNDSGSAGLGVSVKGNRSKENHADLGIFVKSIINGGAASKDGRLRVNDQ 161LIAVNGESLLGKTNQDAMETLRRSMSTEGNKRGMIQLIVA PAR6 2613011 1LPETHRRVRbHKHGSDRPLGFYIRDGMSVRVAPQGLERVPGIFISRLVRGGLAESTGLLAVSD 162EILEVNGIEVAGKTLDQVTDMMVANSHNLIVTVKPANQR PAR6 GAMMA 13537118 1IDVDLVPETHRRVRLHRHGCEKPLGFYIRDGASVRVTPHGLEKVPGIFISRMVPGGLAESTGL 163LAVNDEVLEVNGIEVAGKTLDQVTDMMIANSHNLIVTVKPANQRNNVV PDZ-73 5031978 1RSRKLKEVRLDRLHPEGLGLSVRGGLEFGCGLFISHLIKGGQADSVGLQVGDEIVRINGYSIS 164SCTHEEVINLIRTKKTVSIKVRHIGLIPVKSSPDEFH PDZ-73 5031978 2IPGNRENKEKKVFISLVGSRGLGCSISSGPIQKPGIFISHVKPGSLSAEVGLEIGDQIVNGVD 165FSNLDHKEAVNVLKSSRSLTISIVAAAGRELFMTDEF PDZ-73 5031978 3PEQIMGKDVRLLRIKKEGSLDLALEGGVDSPIGKVVVSAVYERGAAERHGGIVKGDEMAINGK 166IVTDYTLAEADAALQKAWNQGGDVVIDLWAVCPPKEYDD PDZK1 2944188 1LTSTFNPRECKLSKQEGQNYGPFLRIEKDTEGHLVRVVEKCSPAEKAGLQDGDRVLRINGVFV 167DKEEHMQVVDLVRKSGNSVTLLVLDGDSYEKAGSPGIHRD PDZK1 2944188 2RLCYLVKEGGSYGFSLKTVQGKKGVYMTDITPQGVAMRAGVLADDHLIEVNGENVEDASHEEV 168VEKVKKSGSRVMFLLVDKETDKREFIVTD PDZK1 2944188 3QFKRETASLKLLPHQPRIVEMKKGSNGYGFYLRAGSEQKGQIIKDIDSGSPAEEAGLKNNDLV 169VAVNGESVETLDHDSVVEMIRKGGDQTSLLWDKETDNMYRLAEFIVTD PDZK1 2944188 4PDTTEEVDHKPKLCRLAKGENGYGFHLNAIRGLPGSFIKEVQKGGPADLAGLEDEDVIIEVNG 170VNVLDEPYEKVVDRIQSSGKNVTLLVZGKNSS PICK1 4678411 1PTVPGKVTLQKDAQNLIGISIGGGAQYCPCLYIVQVFDNTPAALDGTVAAGDEITGVNRSIKG 171KTKVEVAKMIQEVKGEVTIHYNKLQ PIST 98374330 1SQGVGPIRKVLLLKEDHEGLGISITGGKEHGVPILISEIHPGQPADRCGGLHVGDAILAVNGV 172NLRDTKHKEAVTILSQQRGEIEFEVVYVAPEVDSD prIL16 1478492 1IHVTILHKEEGAGLGFSLAGGADLENKVITVHRVFPNGLASQEGTIQKGNEVLSINGKSLKGT 173THHDALAILRQAREPRQAVIVTRKLTPEEFIVTD prIL16 1478492 2TAEATVCTVTLEKMSAGLGFSLEGGKGSLHGDKPLTINRIFKGAASEQSETVQPGDEILQLGG 174TAMQGLTRFEAWNIIKALPDGPVTIVIRRKSLQSK PSD95 3318652 1LEYEeITLERGNSGLGFSIAGGTDNPHIGDDPSIFITKIIPGGAAAQDGRLRVNDSILFVNEV 175DVREVTHSAAVEALKEAGSIVRLYVMRRKPPAENSS PSD95 3318652 2HVMRRKPPAEKVMEIKLIKGPKGLGFSIAGGVGNQHIPGDNSIYVTKIIEGGAAHKDGRLQIG 176DKILAVNSVGLEDVMHEDAVAALKNTYDVVYLKVAKPSNAYL PSD95 3318652 3REDIPREPRRIVIHRGSTGLGFNIVGGEDGEGIFISFILAGGPADLSGELRKGDQILSVNGVD 177LRNASHEQAAIALKNAGQTVTIIAQYKPEFIVTD PTN-3 179912 1LIRITPDEDGKFGFNLKGGVDQKMPLVVSRINPESPADTCIPKLNEGDQIVLINGRDISEHTH 178DQVVMFIKASRESHSRELALVIRRR PTN-4 190747 1IRMKPDENGRFGFNVKGGYDQKMPVIVSRVAPGTPADLCVPRLNEGDQWLINGRDIAEHTHDQ 179WLFIKASCERHSGELMLLVRPNA PTPL1 515030 1PEREITLVNLKKDAKYGLGFQIIGGEKMGRLDLGIFISSVAPGGPADFHGCLKPGDRLISVNS 180VSLEGVSHHAAIEILQNAPEDVTLVISQPKEKISKVPSTPVHL PTPL1 515030 2GDIFEVELAKNDNSLGISVTGGVNTSVRHGGIYVKAVIPQGAAESDGRIHKGDRVLAVNGVSL 181EGATHKQAVETLRNTGQVVHLLLEKGQSPTSK PTPL1 515030 3TEENTFEVKLFKNSSGLGFSFSREDNLIPEQINASIVRVKKLFAGQPAAESGKIDVGDVILKV 182NGASLKGLSQQEVISALRGTAPEVFLLLCRPPPGVLPEIDT PTPL1 515030 4ELEVELLITLIKSEKASLGFTVTKGNQRIGCYVHDVIQDPAKSDGRLKPGDRLIKVNDTDVTN 183MTHTDAVNLLRAASKTVRLVIGRVLELPRIPMLPH PTPL1 515030 5MLPHLLPDITLTCNKEELGFSLCGGHDSLYQVVYISDINPRSVAAIEGNLQLLDVIHYVNGVS 184TQGMTLEEVNRALDMSLPSLVLKATRNDLPV RGS12 3290015 1RPSPPRVRSVEVARGRAGYGFTLSGQAPCVLSCVMRGSPADFVGLRAGDQILAVNEINVKKAS 185HEDVVKLIGKCSGVLHMVIAEGVGRFESCS RGS3 18644735 1LCSERRYRQITIPRGKDGFGFTICCDSPVRVQAVDSGGPAERAGLQQLDTVLQLNERPVEHWK 186CVELAHEIRSCPSEIILLVWRMVPQVKPGIHRD Rhophilin-like 14279408 1ISFSANKRWTPPRSIRFTAEEGDLGFTLRGNAPVQVHFLDPYCSASVAGAREGDYIVSIQLVD 187CKWLTLSEVMKLLKSFGEDEIEMKVVSLLDSTSSMHNKSAT Serine 2738914 1RGEKKNSSSGISGSQRRYIGVMMLTLSPSILAELQLREPSFPDVQHGVLIHKVILGSPAHRAG 188Protease LRPGDVILAIGEQMVQNAEDVYEAVRTQSQLAVQIRRGRETLTLYV Shank 1 60491851 EEKTVVLQKKDNEGFGFVLRGAKADTPIEEFTPTPAFPALQYLESVDEGGVAWQAGLRTGDFL 189IEVNNENVVKVGHRQVVNMIRQGGNHLVLKVVTVTRNLDPDDTARKKA Shank 3 * 1DYVIDDKVAVLQKRDHEGFGFWRGAKAETPIEEFTPTPAFPALQYLESVDVEGVARAGLRTGD 190LIEVNGVNVVKVGHKQVVALIRQGGNRLVMKVVSVTRKPEEDG Shroom 18652858 1IYLEAFLEGGAPWGFTLKGGLEHGEPLIISKVEEGGKADTLSSKLQAGDEVVHINEVTLSSSR 191KEAVSLVKGSYKTLRLVVRRDVCTDPGH SIP1 2047327 1IRLCRLVRGEQGYGFHLHGEKGRRGQFIRRVEPGSPAEAAALRAGDRLVEVNGVNVEGETHH 192QRIKAVEGQTRLLVVDQN SIP1 2047327 2IRHLRKGPQGYGFNLHSDKSRPGQYIRSVDPGSPAARSGLRAQDRLIEVNGQNVEGLRHAEWA 193IKAREDEARLLVVDPETDE SITAC-18 8886071 1PGVREIHLCKDERGKTGLRLRKVDQGLFVQLVQANTPASLVGLRFGDQLLQIDGRDCAGWSSH 194KAHQVVKKASGQKIVVVNRDRPFQRTVTM SITAC-18 8886071 2PFQRTVTMHKDSMGHVGFVIKKGKIVSLVKGSSAARNGLLTNHYVCEVDGQNVIGLKDKKIME 195ILATAGNVVTLTIIPSVIYEHIVEFIV SSTRIP 7025450 1LKEKTVLLQKKDSEGFGPVLRGAKAQTPIEEFTPTPAFPALQYLESVDEGGVAWRAGLRMGDF 196LIEVNGQNVVKVGHRQVVNMIRQGGNTLMVKVVMVTRHPDMDEAVQ SYNTENIN 2795862 1LEIKQGIREVILCKDQDGKIGLRLKSIDNGIFVQLVQANSPASLVGLRFGDQVLQINGENCAG 197WSSDKAHKVLKQAFGEKITMRIHRD SYNTENIN 2795862 2RDRPFERTITMHKDSTGHVGFIFKNGKITSIVKDSSAARNGLLTEHNICEINGQNVIGLKDSQ 198IADILSTSGNSS Syntrophin 1 1145727 1RRRVTVRKADAGGLGISIKGGRENKMPILISKIFKGLAADQTEALFVGDAILSVNGEDLSSAT 199alpha HDEAVQVLKKTGKEWLEVKYMKDVSPYFK Syntrophin 476700 1IRWKQEAGGLGISIKGGRENRMPILISKIFPGLAADQSRALRLGDAILSVNGTDLRQAHDQAV 200 beta2 OALKRAGKEVLLEVKFIREPIVTD Syntrophin 9507162 1EPFYSGERTVTIRRQTVGGFGLSIKGGAEHNIPVVVSKISKEQRAELSGLLFIGDAILQINGI 201gamma 1 NVVRKCRHEEVVQVLRNAGEEVTLTVSFLKRAPAFLKLP Syntrophin 9507164 1SHQGRNRRTVTLRRQPVGGLGLSIKGGSEHNVPWISKIFEDQAADQTGMLFVGDAVLQVNGIH 202gamma 2 VIHVENATHEEVVHLLRNAGDEVTITVEYLREAPAFLK TAX2-like 3253116 1RGETKEVEVTKTEDALGLTITDNGAGYAFIKRIKEGSIINRIEAVCVGDSIEAINDHSIVGCR 203protein HYEVAKMLRELPKSQPFTLRLVQPKRAF TIAM 1 4507500 1HSIHIEKSDTAADTYGFSLSSVEEDGIRRLYVNSVKETGLASKKGLKAGDEILEINNRAADAL 204NSSMLKDFLSQPSLGLLVRTYPELE TIAM 2 6912703 1PLNVYDVQLTKTGSVCDFGFAVTAQVDERQHLSRIFISDVLPDGLAYGEGLRKGNEIMTLNGE 205AVSDLDLKQMEALFSEKSVGLTLIARPPDTKATL TIP1 2613001 1QRVEIHKLRQGENLILGFSIGGGIDQDPSQNPFSEDKTDKGIYVTRVSEGGPAEIAGLQIGDKI 206MQVNGWDMTMVTHDQARKRLTKRSEEVVRLLVTRQSLQK TIP2 2613003 1RKEVEVFKSEDALGLTITDNGAGYAFIKRIKEGSVIDHIHLISVGDMIEAINGQSLLGCRHYE 207VARLLKELPRGRTFTLKLTEPRK TIP33 2613007 1HSHPRVVELPKTDEGLGFNVMGGKEQNSPIYISRIIPGGVAERHGGLKRGDQLLSVNGVSVEG 208EHHEKAVELLKAAKDSVKLWRYTPKVL TIP43 2613011 1ISNQKRGVKVLKQELGGLGISIKGGKENKMPILISKIFKGLAADQTQALYVGDAILSVNGADL 209RDATHDEAVQALKRAGKEVLLEVKYMREATPYV X-11 beta 3005550 1IHFSNSENCKELQLEKHKGEILGVVVVESGWGSILPTVILANMMNGGPMRSGKLSIGDQIMSI 210NTSLVGLPLATCQGIIKGLKNQTQVKLNIVSCPPVTTVLIKRNSS X-11 beta 3005559 2IPPVTTVLIKRPDLKYQLGFSVQNGIICSLMRGGIAERGGVRVGHRIIEINGQSVVATAHEKI 211VQALSNSVGEIHMKTMPAAMFRLLTGQENSS ZO-1 292937 1IWEQHTVTLHRAPGFGFGIAISGGRDNPHFQSGETSIVISDVLKGGPAEGQLQENDRVAMVNG 212VSMDNVEHAPAVQQLRKSGKNAKITIRRKKKVQIPNSS ZO-1 292937 2ISSQPAKPTKVTLVKSRKNEEYGLRLASHIFVKEISQDSLAARDGNIQEGDVVLKINGTENMS 213STDAKTLIERSKGKLKMVVQRDRATLLNSS ZO-1 292937 3IRMKLVKFRKGDSVGLRLAGGNDVGIFVAGVLEDSPAAKEGLEEGDQILRVNNVDFTNIIREE 214AVLFLLDLPKGEEVTILAQKKKDVFSN ZO-2 12734763 1LIWEQYTVTLQKDSKRGFGIAVSGGRDNPHFENGETSIVISDVLPGGPADGLLQENDRVVMVN 215GTPMEDVLHSFAVQQLRKSGKVAAIVVKRPRKV ZO-2 12734763 2RVLLMKSRANEEYGLRLGSQIFVKEMTRTGLATKDGNLHEGDIILKINGTVTENMSLTDARKL 216IEKSRGKLQLVVLRDS ZO-2 12734763 3HAPNTKMVRFKKGDSVGLRLAGGNDVGIFVAGIQEGTSAEQEGLQEGDQILKVNTQDFRGLVR 217EDAVLYLLEIPKGEMVTILAQSRADVY ZO-3 10092690 1IPGNSTIWEQHTATLSKDPRRGFGIAISGGRDRPGGSMVVSDVVPGGPAEGRLQTGDHIVMVN 218GVSMENATSAFAIQILKTCTKMANITVKRPRRIHLPAEFIVTD ZO-3 10092690 2DVQMKPVKSVLVKRRDSEEFGVKLGSQIFIKHITDSGLAARHRGLQEGDLILQINGVSQNLSL 219NDTRRLIEKSEGKLSLLVLRDRGQFLVNIPNSS ZO-3 10092690 3RGYSPDTRVVRFLKGKSIGLRLAGGNOVGIFVSGVQAGSPADGQGIQEGDQILQVNDVPFQNL 220TFEEAVQFLLGLPPGEEMELVTQRKQDIFWKMVQSEFIVTD

The amino acid sequences provided in Table 2 above may contain aminoacids derived from a fusion protein, e.g., GST. PDZ domain sequence ofparticular interest may be up to 20 amino acids shorter (e.g., 5, 8, 10,12 or 15 amino acids shorter) than the sequence provided in Table 2. Forexample, a sequence may be shortened by up to 3, 6, 9, or 12 amino acidsfrom the C-terminus, the N-terminus, or both termini.

B. Identification of Candidate PL Proteins and Synthesis of Peptides

Certain PDZ domains are bound by the C-terminal residues of PDZ-bindingproteins. To identify PL proteins the C-terminal residues of sequenceswere visually inspected for sequences that one might predict would bindto PDZ-domain containing proteins (see, e.g., Doyle et al., 1996, Cell85, 1067; Songyang et al., 1997, Science 275, 73), including theadditional consensus for PLs identified at Arbor Vita Corporation (U.S.Patent Application 60/360061). TABLE 3 lists some of these proteins, andprovides corresponding C-terminal sequences.

Synthetic peptides of defined sequence (e.g., corresponding to thecarboxyl-termini of the indicated proteins) can be synthesized by anystandard resin-based method (see, e.g., U.S. Pat. No. 4,108,846; seealso, Caruthers et al., 1980, Nucleic Acids Res. Symp. Ser., 215-223;Horn et al., 1980, Nucleic Acids Res. Symp. Ser., 225-232; Roberge, etal., 1995, Science 269:202). The peptides used in the assays describedherein were prepared by the FMOC (see, e.g., Guy and Fields, 1997, Meth.Enz. 289:67-83; Wellings and Atherton, 1997, Meth. Enz. 289:44-67). Insome cases (e.g., for use in the A and G assays of the invention),peptides were labeled with biotin at the amino-terminus by reaction witha four-fold excess of biotin methyl ester in dimethylsulfoxide with acatalytic amount of base. The peptides were cleaved from the resin usinga halide containing acid (e.g. trifluoroacetic acid) in the presence ofappropriate antioxidants (e.g. ethanedithiol) and excess solventlyophilized.

Following lyophilization, peptides can be redissolved and purified byreverse phase high performance liquid chromatography (HPLC). Oneappropriate HPLC solvent system involves a Vydac C-18 semi-preparativecolumn running at 5 mL per minute with increasing quantities ofacetonitrile plus 0.1% trifluoroacetic acid in a base solvent of waterplus 0.1% trifluoroacetic acid. After HPLC purification, the identitiesof the peptides are confirmed by MALDI cation-mode mass spectrometry.

C. Detecting PDZ-PL Interactions

The present inventors were able in part to identify the interactionssummarized in TABLE 4 by developing new high throughput screening assayswhich are described in greater detail infra. Various other assay formatsknown in the art can be used to select ligands that are specificallyreactive with a particular protein. For example, solid-phase ELISAimmunoassays, immunoprecipitation, Biacore, and Western blot assays canbe used to identify peptides that specifically bind PDZ-domainpolypeptides. As discussed supra, two different, complementary assayswere developed to detect PDZ-PL interactions. In each, one bindingpartner of a PDZ-PL pair is immobilized, and the ability of the secondbinding partner to bind is determined. These assays, which are describedinfra, can be readily used to screen for hundreds to thousands ofpotential PDZ-ligand interactions in a few hours. Thus these assays canbe used to identify yet more novel PDZ-PL interactions in cells. Inaddition, they can be used to identify antagonists of PDZ-PLinteractions (see infra).

In various embodiments, fusion proteins are used in the assays anddevices of the invention. Methods for constructing and expressing fusionproteins are well known. Fusion proteins generally are described inAusubel et al., supra, Kroll et al., 1993, DNA Cell. Biol. 12:441, andImai et al., 1997, Cell 91:521-30. Usually, the fusion protein includesa domain to facilitate immobilization of the protein to a solidsubstrate (“an immobilization domain”). Often, the immobilization domainincludes an epitope tag (i.e., a sequence recognized by an antibody,typically a monoclonal antibody) such as polyhistidine (Bush et al,1991, J. Biol Chem 266:13811-14), SEAP (Berger et al, 1988, Gene66:1-10), or M1 and M2 flag (see, e.g., U.S. Pat. Nos. 5,011,912;4,851,341; 4,703,004; 4,782,137). In an embodiment, the immobilizationdomain is a GST coding region. It will be recognized that, in additionto the PDZ-domain and the particular residues bound by an immobilizedantibody, protein A, or otherwise contacted with the surface, theprotein (e.g., fusion protein), will contain additional residues. Insome embodiments these are residues naturally associated with thePDZ-domain (i.e., in a particular PDZ-protein) but they may includeresidues of synthetic (e.g., poly(alanine)) or heterologous origin(e.g., spacers of, e.g., between 10 and 300 residues).

PDZ domain-containing polypeptide used in the methods of the invention(e.g., PDZ fusion proteins) of the invention are typically made by (1)constructing a vector (e.g., plasmid, phage or phagemid) comprising apolynucleotide sequence encoding the desired polypeptide, (2)introducing the vector into an suitable expression system (e.g., aprokaryotic, insect, mammalian, or cell free expression system), (3)expressing the fusion protein and (4) optionally purifying the fusionprotein.

(1) In one embodiment, expression of the protein comprises inserting thecoding sequence into an appropriate expression vector (i.e., a vectorthat contains the necessary elements for the transcription andtranslation of the inserted coding sequence required for the expressionsystem employed, e.g., control elements including enhancers, promoters;transcription terminators, origins of replication, a suitable initiationcodon (e.g., methionine), open reading frame, and translationalregulatory signals (e.g., a ribosome binding site, a termination codonand a polyadenylation sequence. Depending on the vector system and hostutilized, any number of suitable transcription and translation elements,including constitutive and inducible promoters, can be used.

The coding sequence of the fusion protein includes a PDZ domain and animmobilization domain as described elsewhere herein. Polynucleotidesencoding the amino acid sequence for each domain can be obtained in avariety of ways known in the art; typically the polynucleotides areobtained by PCR amplification of cloned plasmids, cDNA libraries, andcDNA generated by reverse transcription of RNA, using primers designedbased on sequences determined by the practitioner or, more often,publicly available (e.g., through GenBank). The primers include linkerregions (e.g., sequences including restriction sites) to facilitatecloning and manipulation in production of the fusion construct. Thepolynucleotides corresponding to the PDZ and immobilization regions arejoined in-frame to produce the fusion protein-encoding sequence.

The fusion proteins of the invention may be expressed as secretedproteins (e.g., by including the signal sequence encoding DNA in thefusion gene; see, e.g., Lui et al, 1993, PNAS USA, 90:8957-61) or asnonsecreted proteins.

In some embodiments, the PDZ-containing proteins or PL polypeptides areimmobilized on a solid surface. The substrate to which the polypeptideis bound may in any of a variety of forms, e.g., a microtiter dish, atest tube, a dipstick, a microcentrifuge tube, a bead, a spinnable disk,a permeable or semi-permeable membrane, and the like. Suitable materialsinclude glass, plastic (e.g., polyethylene, PVC, polypropylene,polystyrene, and the like), protein, paper, carbohydrate, lipidmonolayer or supported lipid bilayer, films and other solid supports.Other materials that may be employed include ceramics, metals,metalloids, semiconductive materials, cements and the like.

In some embodiments, the PDZ and/or PL fusion proteins are organized asan array. The term “array,” as used herein, refers to an orderedarrangement of immobilized fusion proteins, in which particulardifferent fusion proteins (i.e., having different PDZ domains) arelocated at different predetermined sites on the substrate. Because thelocation of particular fusion proteins on the array is known, binding atthat location can be correlated with binding to the PDZ domain situatedat that location. Immobilization of fusion proteins on beads(individually or in groups) is another particularly useful approach. Inone embodiment, individual fusion proteins are immobilized on beads. Inone embodiment, mixtures of distinguishable beads are used.Distinguishable beads are beads that can be separated from each other onthe basis of a property such as size, magnetic property, color (e.g.,using FACS) or affinity tag (e.g., a bead coated with protein A can beseparated from a bead not coated with protein A by using IgG affinitymethods). Binding to particular PDZ domain may be determined.

Methods for immobilizing proteins are known, and include covalent andnon-covalent methods. One suitable immobilization method isantibody-mediated immobilization. According to this method, an antibodyspecific for the sequence of an “immobilization domain” of thePDZ-domain containing protein is itself immobilized on the substrate(e.g., by adsorption). One advantage of this approach is that a singleantibody may be adhered to the substrate and used for immobilization ofa number of polypeptides (sharing the same immobilization domain). Forexample, an immobilization domain consisting of poly-histidine (Bush etal, 1991, J. Biol Chem 2 266: 13811-14) can be bound by ananti-histidine monoclonal antibody (R&D Systems, Minneapolis, Minn.); animmobilization domain consisting of secreted alkaline phosphatase(“SEAP”) (Berger et al, 1988, Gene 66:1-10) can be bound by anti-SEAP(Sigma Chemical Company, St. Louis, Mo.); an immobilization domainconsisting of a FLAG epitope can be bound by anti-FLAG. Otherligand-antiligand immobilization methods are also suitable (e.g., animmobilization domain consisting of protein A sequences (Harlow andLane, 1988, Antibodies A Laboratory Manual, Cold Spring HarborLaboratory; Sigma Chemical Co., St. Louis, Mo.) can be bound by IgG; andan immobilization domain consisting of streptavidin can be bound bybiotin (Harlow & Lane, supra; Sigma Chemical Co., St. Louis, Mo.). In apreferred embodiment, the immobilization domain is a GST moiety, asdescribed herein.

When antibody-mediated immobilization methods are used, glass andplastic are especially useful substrates. The substrates may be printedwith a hydrophobic (e.g., Teflon) mask to form wells. Preprinted glassslides with 3, 10 and 21 wells per 14.5 cm² slide “working area” areavailable from, e.g., SPI Supplies, West Chester, Pa.; also see U.S.Pat. No. 4,011,350). In certain applications, a large format (12.4cm×8.3 cm) glass slide is printed in a 96 well format is used; thisformat facilitates the use of automated liquid handling equipment andutilization of 96 well format plate readers of various types(fluorescent, calorimetric, scintillation). However, higher densitiesmay be used (e.g., more than 10 or 100 polypeptides per cm²). See, e.g.,MacBeath et al, 2000, Science 289:1760-63.

Typically, antibodies are bound to substrates (e.g., glass substrates)by adsorption. Suitable adsorption conditions are well known in the artand include incubation of 0.5-50 ug/ml (e.g., 10 ug/ml) mAb in buffer(e.g., PBS, or 50 to 300 mM Tris, MOPS, HEPES, PIPES, acetate buffers,pHs 6.5 to 8, at 4° C.) to 37° C. and from 1 hr to more than 24 hours.

Proteins may be covalently bound or noncovalently attached throughnonspecific bonding. If covalent bonding between the fusion protein andthe surface is desired, the surface will usually be polyfunctional or becapable of being polyfunctionalized. Functional groups which may bepresent on the surface and used for linking can include carboxylicacids, aldehydes, amino groups, cyano groups, ethylenic groups, hydroxylgroups, mercapto groups and the like. The manner of linking a widevariety of compounds to various surfaces is well known and is amplyillustrated in the literature.

Exemplary assays are provided below.

“A Assay” Detection of PDZ-Ligand Binding Using Immobilized PL Peptide.

In one aspect, the invention provides an assay in which biotinylatedcandidate PL peptides are immobilized on an avidin-coated surface. Thebinding of PDZ-domain fusion protein to this surface is then measured.In a preferred embodiment, the PDZ-domain fusion protein is a GST/PDZfusion protein and the assay is carried out as follows:

(1) Avidin is bound to a surface, e.g. a protein binding surface. In oneembodiment, avidin is bound to a polystyrene 96 well plate (e.g., NuncPolysorb (cat #475094) by addition of 100 uL per well of 20 ug/mL ofavidin (Pierce) in phosphate buffered saline without calcium andmagnesium, pH 7.4 (“PBS”, GibcoBRL) at 4° C. for 12 hours. The plate isthen treated to block nonspecific interactions by addition of 200 uL perwell of PBS containing 2 g per 100 mL protease-free bovine serum albumin(“PBS/BSA”) for 2 hours at 4° C. The plate is then washed 3 times withPBS by repeatedly adding 200 uL per well of PBS to each well of the,plate and then dumping the contents of the plate into a waste containerand tapping the plate gently on a dry surface.

(2) Biotinylated PL peptides (or candidate PL peptides, e.g. see TABLE3) are immobilized on the surface of wells of the plate by addition of50 uL per well of 0.4 uM peptide in PBS/BSA for 30 minutes at 4° C.Usually, each different peptide is added to at least eight differentwells so that multiple measurements (e.g. duplicates and alsomeasurements using different (GST/PDZ-domain fusion proteins and a GSTalone negative control) can be made, and also additional negativecontrol wells are prepared in which no peptide is immobilized. Followingimmobilization of the PL peptide on the surface, the plate is washed 3times with PBS.

(3) GST/PDZ-domain fusion protein (prepared as described supra) isallowed to react with the surface by addition of 50 uL per well of asolution containing 5 ug/mL GST/PDZ-domain fusion protein in PBS/BSA for2 hours at 4° C. As a negative control, GST alone (i.e. not a fusionprotein) is added to specified wells, generally at least 2 wells (i.e.duplicate measurements) for each immobilized peptide. After the 2 hourreaction, the plate is washed 3 times with PBS to remove unbound fusionprotein.

(4) The binding of the GST/PDZ-domain fusion protein to theavidin-biotinylated peptide surface can be detected using a variety ofmethods, and detectors known in the art. In one embodiment, 50 uL perwell of an anti-GST antibody in PBS/BSA (e.g. 2.5 ug/mL of polyclonalgoat-anti-GST antibody, Pierce) is added to the plate and allowed toreact for 20 minutes at 4° C. The plate is washed 3 times with PBS and asecond, detectably labeled antibody is added. In one embodiment, 50 uLper well of 2.5 ug/mL of horseradish peroxidase (HRP)-conjugatedpolyclonal rabbit anti-goat immunoglobulin antibody is added to theplate and allowed to react for 20 minutes at 4° C. The plate is washed 5times with 50 mM Tris pH 8.0 containing 0.2% Tween 20, and developed byaddition of 100 uL per well of HRP-substrate solution (TMB, Dako) for 20minutes at room temperature (RT). The reaction of the HRP and itssubstrate is terminated by the addition of 100 uL per well of 1Msulfuric acid and the absorbance (A) of each well of the plate is readat 450 nm.

(5) Specific binding of a PL peptide and a PDZ-domain polypeptide isdetected by comparing the signal from the well(s) in which the PLpeptide and PDZ domain polypeptide are combined with the backgroundsignal(s). The background signal is the signal found in the negativecontrols. Typically a specific or selective reaction will be at leasttwice background signal, more typically more than 5 times background,and most typically 10 or more times the background signal. In addition,a statistically significant reaction will involve multiple measurementsof the reaction with the signal and the background differing by at leasttwo standard errors, more typically four standard errors, and mosttypically six or more standard errors. Correspondingly, a statisticaltest (e.g. a T-test) comparing repeated measurements of the signal withrepeated measurements of the background will result in a p-value <0.05,more typically a p-value <0.01, and most typically a p-value <0.001 orless.

As noted, in an embodiment of the “A” assay, the signal from binding ofa GST/PDZ-domain fusion protein to an avidin surface not exposed to(i.e. not covered with) the PL peptide is one suitable negative control(sometimes referred to as “B”). The signal from binding of GSTpolypeptide alone (i.e. not a fusion protein) to an avidin-coatedsurface that has been exposed to (i.e. covered with) the PL peptide is asecond suitable negative control (sometimes referred to as “B2”).Because all measurements are done in multiples (i.e. at least duplicate)the arithmetic mean (or, equivalently, average) of several measurementsis used in determining the binding, and the standard error of the meanis used in determining the probable error in the measurement of thebinding. The standard error of the mean of N measurements equals thesquare root of the following: the sum of the squares of the differencebetween each measurement and the mean, divided by the product of (N) and(N−1). Thus, in one embodiment, specific binding of the PDZ protein tothe plate-bound PL peptide is determined by comparing the mean signal(“mean S”) and standard error of the signal (“SE”) for a particularPL-PDZ combination with the mean B1 and/or mean B2.

“G Assay”—Detection of PDZ-Ligand Binding Using Immobilized PDZ-DomainFusion Polypeptide

In one aspect, the invention provides an assay in which a GST/PDZ fusionprotein is immobilized on a surface (“G” assay). The binding of labeledPL peptide (e.g., as listed in TABLE 3) to this surface is thenmeasured. In a preferred embodiment, the assay is carried out asfollows:

(1) A PDZ-domain polypeptide is bound to a surface, e.g. a proteinbinding surface. In a preferred embodiment, a GST/PDZ fusion proteincontaining one or more PDZ domains is bound to a polystyrene 96-wellplate. The GST/PDZ fusion protein can be bound to the plate by any of avariety of standard methods known to one of skill in the art, althoughsome care must be taken that the process of binding the fusion proteinto the plate does not alter the ligand-binding properties of the PDZdomain. In one embodiment, the GST/PDZ fusion protein is bound via ananti-GST antibody that is coated onto the 96-well plate. Adequatebinding to the plate can be achieved when:

a. 100 uL per well of 5 ug/mL goat anti-GST polyclonal antibody (Pierce)in PBS is added to a polystyrene 96-well plate (e.g., Nunc Polysorb) at4° C. for 12 hours.

b. The plate is blocked by addition of 200 uL per well of PBS/BSA for 2hours at 4° C.

c. The plate is washed 3 times with PBS.

d. 50 uL per well of 5 ug/mL GST/PDZ fusion protein) or, as a negativecontrol, GST polypeptide alone (i.e. not a fusion protein) in PBS/BSA isadded to the plate for 2 hours at 4° C.

e. The plate is again washed 3 times with PBS.

(2) Biotinylated PL peptides are allowed to react with the surface byaddition of 50 uL per well of 20 uM solution of the biotinylated peptidein PBS/BSA for 10 minutes at 4° C., followed by an additional 20 minuteincubation at 25° C. The plate is washed 3 times with ice cold PBS.

(3) The binding of the biotinylated peptide to the GST/PDZ fusionprotein surface can be detected using a variety of methods and detectorsknown to one of skill in the art. In one embodiment, 100 uL per well of0.5 ug/mL streptavidin-horse radish peroxidase (HRP) conjugate dissolvedin BSA/PBS is added and allowed to react for 20 minutes at 4° C. Theplate is then washed 5 times with 50 mM Tris pH 8.0 containing 0.2%Tween 20, and developed by addition of 100 uL per well of HRP-substratesolution (TMB, Dako) for 20 minutes at room temperature (RT). Thereaction of the HRP and its substrate is terminated by addition of 100uL per well of 1M sulfuric acid, and the absorbance of each well of theplate is read at 450 nm.

(4) Specific binding of a PL peptide and a PDZ domain polypeptide isdetermined by comparing the signal from the well(s) in which the PLpeptide and PDZ domain polypeptide are combined, with the backgroundsignal(s). The background signal is the signal found in the negativecontrol(s). Typically a specific or selective reaction will be at leasttwice background signal, more typically more than 5 times background,and most typically 10 or more times the background signal. In addition,a statistically significant reaction will involve multiple measurementsof the reaction with the signal and the background differing by at leasttwo standard errors, more typically four standard errors, and mosttypically six or more standard errors. Correspondingly, a statisticaltest (e.g. a T-test) comparing repeated measurements of the signal withrepeated measurements of the background will result in a p-value <0.05,more typically a p-value <0.01, and most typically a p-value <0.001 orless. As noted, in an embodiment of the “G” assay, the signal frombinding of a given PL peptide to immobilized (surface bound) GSTpolypeptide alone is one suitable negative control (sometimes referredto as “B 1”). Because all measurement are done in multiples (i.e. atleast duplicate) the arithmetic mean (or, equivalently, average.) ofseveral measurements is used in determining the binding, and thestandard error of the mean is used in determining the probable error inthe measurement of the binding. The standard error of the mean of Nmeasurements equals the square root of the following: the sum of thesquares of the difference between each measurement and the mean, dividedby the product of (N) and (N−1). Thus, in one embodiment, specificbinding of the PDZ protein to the platebound peptide is determined bycomparing the mean signal (“mean S”) and standard error of the signal(“SE”) for a particular PL-PDZ combination with the mean B1.

“G′ Assay” and “G” Assay”

Two specific modifications of the specific conditions described suprafor the “G assay” are particularly useful. The modified assays uselesser quantities of labeled PL peptide and have slightly differentbiochemical requirements for detection of PDZ-ligand binding compared tothe specific assay conditions described supra.

For convenience, the assay conditions described in this section arereferred to as the “G′ assay” and the “G” assay,” with the specificconditions described in the preceding section on G assays being referredto as the “G⁰ assay.” The “G′ assay” is identical to the “G⁰ assay”except at step (2) the peptide concentration is 10 uM instead of 20 uM.This results in slightly lower sensitivity for detection of interactionswith low affinity and/or rapid dissociation rate. Correspondingly, itslightly increases the certainty that detected interactions are ofsufficient affinity and half-life to be of biological importance anduseful therapeutic targets.

The “G” assay” is identical to the “G⁰ assay” except that at step (2)the peptide concentration is 1 uM instead of 20 uM and the incubation isperformed for 60 minutes at 25° C. (rather than, e.g., 10 minutes at 4°C. followed by 20 minutes at 25° C.). This results in lower sensitivityfor interactions of low affinity, rapid dissociation rate, and/oraffinity that is less at 25° C. than at 4° C. Interactions will havelower affinity at 25° C. than at 4° C. if (as we have found to begenerally true for PDZ-ligand binding) the reaction entropy is negative(i.e. the entropy of the products is less than the entropy of thereactants). In contrast, the PDZ-PL binding signal may be similar in the“G” assay” and the “G⁰ assay” for interactions of slow association anddissociation rate, as the PDZ-PL complex will accumulate during thelonger incubation of the “G” assay.” Thus comparison of results of the“G” assay” and the “G⁰ assay” can be used to estimate the relativeentropies, enthalpies, and kinetics of different PDZ-PL interactions.(Entropies and enthalpies are related to binding affinity by theequations delta G=RT In (Kd)=delta H-T delta S where delta G, H, and Sare the reaction free energy, enthalpy, and entropy respectively, T isthe temperature in degrees Kelvin, R is the gas constant, and Kd is theequilibrium dissociation constant). In particular, interactions that aredetected only or much more strongly in the “G⁰ assay” generally have arapid dissociation rate at 25° C. (t1/2<10 minutes) and a negativereaction entropy, while interactions that are detected similarlystrongly in the “G” assay” generally have a slower dissociation rate at25° C. (t1/2>10 minutes). Rough estimation of the thermodynamics andkinetics of PDZ-PL interactions (as can be achieved via comparison ofresults of the “G⁰ assay” versus the “G” assay” as outlined supra) canbe used in the design of efficient inhibitors of the interactions. Forexample, a small molecule inhibitor based on the chemical structure of aPL that dissociates slowly from a given PDZ domain (as evidenced bysimilar binding in the “G” assay” as in the “G⁰ assay”) may itselfdissociate slowly and thus be of high affinity.

In this manner, variation of the temperature and duration of step (2) ofthe “G assay” can be used to provide insight into the kinetics andthermodynamics of the PDZ-ligand binding reaction and into design ofinhibitors of the reaction.

Assay Variations

As discussed supra, it will be appreciated that many of the steps in theabove-described assays can be varied, for example, various substratescan be used for binding the PL and PDZ-containing proteins; differenttypes of PDZ containing fusion proteins can be used; different labelsfor detecting PDZ/PL interactions can be employed; and different ways ofdetection can be used.

The PDZ-PL detection assays can employ a variety of surfaces to bind thePL and/or PDZ-containing proteins. For example, a surface can be an“assay plate” which is formed from a material (e.g. polystyrene) whichoptimizes adherence of either the PL protein or PDZ-containing proteinthereto. Generally, the individual wells of the assay plate will have ahigh surface area to volume ratio and therefore a suitable shape is aflat bottom well (where the proteins of the assays are adherent). Othersurfaces include, but are not limited to, polystyrene or glass beads,polystyrene or glass slides, papers, dipsticks, plastics, films and thelike.

For example, the assay plate can be a “microtiter” plate. The term“microtiter” plate when used herein refers to a multiwell assay plate,e.g., having between about 30 to 200 individual wells, usually 96 wells.Alternatively, high-density arrays can be used. Often, the individualwells of the microtiter plate will hold a maximum volume of about 250ul. Conveniently, the assay plate is a 96 well polystyrene plate (suchas that sold by Becton Dickinson Labware, Lincoln Park, N.J.), whichallows for automation and high throughput screening. Other surfacesinclude polystyrene microtiter ELISA plates such as that sold by NuncMaxisorp, Inter Med, Denmark. Often, about 50 ul to 300 ul, morepreferably 100 ul to 200 ul, of an aqueous sample comprising bufferssuspended therein will be added to each well of the assay plate.

The detectable labels of the invention can be any detectable compound orcomposition which is conjugated directly or indirectly with a molecule(such as described above). The label can be detectable by itself (e.g.,radioisotope labels or fluorescent labels) or, in the case of anenzymatic label, can catalyze a chemical alteration of a substratecompound or composition which is detectable. The preferred label is anenzymatic one which catalyzes a color change of a non-radioactive colorreagent.

Sometimes, the label is indirectly conjugated with the antibody. One ofskill is aware of various techniques for direct and indirectconjugation. For example, the antibody can be conjugated with biotin andany of the categories of labels mentioned above can be conjugated withavidin, or vice versa (see also “A” and “G” assay above). Biotin bindsselectively to avidin and thus, the label can be conjugated with theantibody in this indirect manner. See, Ausubel, supra, for a review oftechniques involving biotin-avidin conjugation and similar assays.Alternatively, to achieve indirect conjugation of the label with theantibody, the antibody is conjugated with a small hapten (e.g. digoxin)and one of the different types of labels mentioned above is conjugatedwith an anti-hapten antibody (e.g. anti-digoxin antibody). Thus,indirect conjugation of the label with the antibody can be achieved.

Assay variations can include different washing steps. By “washing” ismeant exposing the solid phase to an aqueous solution (usually a bufferor cell culture media) in such a way that unbound material (e.g.,non-adhering cells, non-adhering capture agent, unbound ligand,receptor, receptor construct, cell lysate, or HRP antibody) is removedtherefrom. To reduce background noise, it is convenient to include adetergent (e.g., Triton X) in the washing solution. Usually, the aqueouswashing solution is decanted from the wells of the assay plate followingwashing. Conveniently, washing can be achieved using an automatedwashing device. Sometimes, several washing steps (e.g., between about 1to 10 washing steps) can be required.

Various buffers can also be used in PDZ-PL detection assays. Forexample, various blocking buffers can be used to reduce assaybackground. The term “blocking buffer” refers to an aqueous, pH bufferedsolution containing at least one blocking compound which is able to bindto exposed surfaces of the substrate which are not coated with a PL orPDZ-containing protein. The blocking compound is normally a protein suchas bovine serum albumin (BSA), gelatin, casein or milk powder and doesnot cross-react with any of the reagents in the assay. The block bufferis generally provided at a pH between about 7 to 7.5 and suitablebuffering agents include phosphate and TRIS.

Various enzyme-substrate combinations can also be utilized in detectingPDZ-PL interactions. Examples of enzyme-substrate combinations include,for example:

(i) Horseradish peroxidase (HRP or HRPO) with hydrogen peroxidase as asubstrate, wherein the hydrogen peroxidase oxidizes a dye precursor(e.g. orthophenylene diamine [OPD] or 3,3′,5,5′-tetramethyl benzidinehydrochloride [TMB]) (as described above).

(ii) alkaline phosphatase (AP) with para-Nitrophenyl phosphate aschromogenic substrate.

(iii) Beta-D-galactosidase (Beta D-Gal) with a chromogenic substrate(e.g. p-nitrophenyl-Beta-D-galactosidase) or fluorogenic substrate4-methylumbelliferyl-Beta-D-galactosidase.

Numerous other enzyme-substrate combinations are available to thoseskilled in the art. For a general review of these, see U.S. Pat. Nos.4,275,149 and 4,318,980, both of which are herein incorporated byreference.

Further, it will be appreciated that, although, for convenience, thepresent discussion primarily refers to detection of PDZ-PL interactions,agonists or antagonists of PDZ-PL interactions can be used to diagnosecellular abnormalities.

V. Collection of Tissue Samples Such as Cervical Tissues

Diagnosing the presence of pathogens requires collection of samplesappropriate to the organism. For detection of oncogenic HPV E6 proteins,one would collect tissue for testing from the cervix, penis, anus, orthroat using a scrape, swab or biopsy technique. For diagnosis ofbloodborne pathogens such as HIV, collection of blood through standardmeans would be most appropriate. Diagnosis of fungal or viral infectionsthat may have caused skin lesions would require the collection of asample from the affected area.

This invention is not intended to cover sampling devices. However, itshould be noted that since the invention is predicated on the detectionof PDZ or PL proteins, appropriate care must be taken to collect asufficient amount of sample to detect pathogen proteins and to maintainthe integrity of proteins in the sample. The amount of sample to collectshould be determined empirically for each diagnostic test. Factors inthe decision may include, but not be limited to, the stage at whichdetection is desired, the amount of pathogen per unit sample, the amountof diagnostic protein per unit per unit sample, availability ofdiagnostic epitopes and the stability of diagnostic epitopes.

Exemplary collection devices for cervical tissue include, but are notlimited to, those described in U.S. Pat. Nos. 6,241,687, 6,352,513,6,336,905, 6,115,990 and 6,346,086. These collection devices wouldfacilitate the collection of cervical tissue for the diagnosis ofoncogenic human papillomavirus infection. These devices arepredominantly collection of cervical cells or tissues through scraping;alternatively, one could use standard biopsy methods to collect samplesfrom any tissues to be examined.

Although the diagnostic method disclosed in this application is directedat the detection of PL proteins, sample collection need not be limitedto collection of proteins. One could alternatively collect RNA fromtissue samples, use an in vitro translation kit to produce protein fromcollected templates, and then assay using methods disclosed herein. In asimilar manner, DNA could be collected from test samples, specificprimers for oncogenic E6 proteins could be used to either amplify theDNA content (using a DNA polymerase) or transcribe and translate thesample into proteins that could be tested with methods disclosed herein.

VI. Assays for Detecting Oncogenic E6 Proteins

Oncogenic E6 proteins can be detected by their ability to bind to PDZdomains. This could be a developed into a single detection stageapproach or more favorably as a two-stage or ‘sandwich’ approach forincreased sensitivity and specificity.

For single stage approaches, a ‘tagged’ version of a PDZ domain thatspecifically recognizes oncogenic E6 proteins, such as those disclosedin TABLES 3 and 4, can be used to directly probe for the presence ofoncogenic E6 protein in a sample. As noted supra, an example of thiswould be to attach the test sample to a solid support (for example,cervical cells or tissue could be coated on a slide and ‘fixed’ topermeablize the cell membranes), incubate the sample with a tagged ‘PLdetector’ protein (a PDZ domain fusion) under appropriate conditions,wash away unbound PL detector, and assay for the presence of the ‘tag’in the sample. In addition, even without a tag, one could measure thephysical properties of the PDZ protein and the PDZ protein bound to andE6 protein. Techniques such as surface plasmon resonance, circulardichoism, and other techniques that directly assess binding could beused to detect the presence of oncogenic E6 proteins. One should note,however, that PDZ domains may also bind endogenous cellular proteins.Thus, frequency of binding must be compared to control cells that do notcontain E6 oncoproteins or the ‘PL detector’ should be modified suchthat it is significantly more specific for the oncogenic E6 proteins(see section X).

For two-stage or sandwich approaches, use of the PL detector is coupledwith a second method of either capturing or detecting captured proteins.The second method could be using an antibody that binds to the E6oncoprotein or a second compound or protein that can bind to E6oncorproteins at a location on the E6 protein that does not reduce theavailability of the E6 PL. Such proteins may include, but not be limitedto, p53, E6-AP, E6-BP or engineered compounds that bind E6 oncoproteins.Alternatively, one could also use DNA binding or Zn2+ binding to assayfor the presence of captured E6 protein, since oncogenic E6 proteins areknown to bind certain DNA structures through the use of divalentcations. Additionally, one could use the PDZ-captured E6 protein in anactivity assay, since E6 is known to degrade DNA and certain proteinsincluding p53 in the presence of a reticulocyte lysate.

Antibodies

Many biological assays are designed as a ‘sandwich’, where an antibodyconstitutes one side of the sandwich. This method can improve the signalto noise ratio for a diagnostic by reducing background signal andamplifying appropriate signals. Antibodies can be generated thatspecifically recognize the diagnostic protein. Since this inventiondiscloses the method of using PDZ or PL proteins to diagnose pathogeninfections, antibodies should be generated that do not conflict with thePDZ:PL interaction.

For the production of antibodies, various host animals, including butnot limited to rabbits, mice, rats, etc., may be immunized by injectionwith a peptide. The peptide may be attached to a suitable carrier, suchas BSA or KLH, by means of a side chain functional group or linkersattached to a side chain functional group. Various adjuvants may be usedto increase the immunological response, depending on the host species,including but not limited to Freund's (complete and incomplete), mineralgels such as aluminum hydroxide, surface active substances such aslysolecithin, pluronic polyols, polyanions, peptides, oil emulsions,keyhole limpet hemocyanin, dinitrophenol, and potentially useful humanadjuvants such as BCG (bacilli Calmette-Guerin) and Corynebacteriumparvum.

Monoclonal antibodies to a peptide may be prepared using any techniquewhich provides for the production of antibody molecules by continuouscell lines in culture. These include but are not limited to thehybridoma technique originally described by Koehler and Milstein, 1975,Nature 256:495-497, the human B-cell hybridoma technique, Kosbor et al.,1983, Immunology Today 4:72; Cote et al., 1983, Proc. Natl. Acad. Sci.U.S.A. 80:2026-2030 and the EBV-hybridoma technique (Cole et al., 1985,Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96(1985)). In addition, techniques developed for the production of“chimeric antibodies” (Morrison et al., 1984, Proc. Natl. Acad. Sci.U.S.A. 81:6851-6855; Neuberger et al., 1984, Nature 312:604-608; Takedaet al., 1985, Nature 314:452-454) by splicing the genes from a mouseantibody molecule of appropriate antigen specificity together with genesfrom a human antibody molecule of appropriate biological activity can beused. Alternatively, techniques described for the production of singlechain antibodies (U.S. Pat. No. 4,946,778) can be adapted to producepeptide-specific single chain antibodies.

Antibody fragments which contain deletions of specific binding sites maybe generated by known techniques. For example, such fragments includebut are not limited to F(ab′)₂ fragments, which can be produced bypepsin digestion of the antibody molecule and Fab fragments, which canbe generated by reducing the disulfide bridges of the F(ab′)₂ fragments.Alternatively, Fab expression libraries may be constructed (Huse et al.,1989, Science 246:1275-1281) to allow rapid and easy identification ofmonoclonal Fab fragments with the desired specificity for the peptide ofinterest.

The antibody or antibody fragment specific for the desired peptide canbe attached, for example, to agarose, and the antibody-agarose complexis used in immunochromatography to purify peptides of the invention.See, Scopes, 1984, Protein Purification: Principles and Practice,Springer-Verlag New York, Inc., NY, Livingstone, 1974, MethodsEnzymology: Immunoaffinity Chromatography of Proteins 34:723-731.Antibodies can also be linked to other solid supports for diagnosticapplications, or alternatively labeled with a means of detection such anenzyme that can cleave a colorimetric substrate, a fluorophore, amagnetic particle, or other measurable compositions of matter.

Specific antibodies against E6 proteins have historically been difficultto produce. In conjunction with the methods describe supra, one couldemploy a number of techniques to increase the likelihood of producing orselecting high affinity antibodies. An example is to prepare the E6antigen (to raise antibodies against) in the same manner that one wouldprepare tissue or cell samples for testing. Alternatively, one couldimmunize with E6 fusion protein prepared in one manner, and screen forspecific E6 antibodies using a second E6 protein prepared in a differentmanner. This should select for antibodies that recognize E6 epitopesthat are conserved under different sample collection and preparationprocedures. In another example, one could immunize animals with E6antigen that has been rapidly denatured and renatured, such thatepitopes that are insensitive to preparation conditions are selectedfor. Another method that could be employed is to use peptidescorresponding to antigenic regions of the E6 proteins as predicted byMajor Histocompatibility Complex (MHC) and T Cell Receptor (TCR)consensus binding.

2. Alternative Detection Methods for Captured E6 Protein

E6 proteins that have been captured by PDZ domains could be detected byseveral alternative methods. Several proteins are known to associatewith E6 proteins. Any of them that had a reasonable affinity for E6could be used to detect the presence of captured and concentrated E6protein in a sample by one skilled in the art. In addition, new bindingproteins or aptamers could be identified that bound to E6 proteins.Third, activity assays specific for E6 could be employed.

The detection assay itself could also be carried out using a variety ofmethods. A standard ELISA using a PDZ to capture could be set up as acompetition, where the PDZ domain is pre-loaded with a labeled PL thathas lower affinity than the E6 proteins. Thus, in the presence of E6,the label is displaced and one sees a reduction of signal thatcorresponds to E6 presence. Other variants that use aspects ofcompetition and inhibition of binding are intended to be included aswell. One variant could even have the PL covalently attached to the PDZdomain through a linker such that the PL could bind it's own PDZ domain.Using donor quenching dyes, one would only see an increase in signalwhen the PL of an oncogenic E6 protein was able to displace the labeledPL. All such competition methods must be measured against controls thatassess the amount of endogenous PL proteins that can bind the PDZ domainused to assess the presence of oncogenic E6 proteins.

VIII. Measurements of Assay Sensitivity

The “A” and “G” assays of the invention can be used to determine the“apparent affinity” of binding of a PDZ ligand peptide to a PDZ-domainpolypeptide. Apparent affinity is determined based on the concentrationof one molecule required to saturate the binding of a second molecule(e.g., the binding of a ligand to a receptor). Two particularly usefulapproaches for quantitation of apparent affinity of PDZ-ligand bindingare provided infra. These methods can be used to compare the sensitivityand affinity of differing PL detector constructs. Understanding thesensitivity of the PDZ for pathogen PLs is essential because it helps todefine the amount of tissue or cell sample that must be tested to obtaina definitive result.

(1) A GST/PDZ fusion protein, as well as GST alone as a negativecontrol, are bound to a surface (e.g., a 96-well plate) and the surfaceblocked and washed as described supra for the “G” assay.

(2) 50 uL per well of a solution of biotinylated PL peptide (e.g. asshown in TABLE 3) is added to the surface in increasing concentrationsin PBS/BSA (e.g. at 0.1 uM, 0.33 uM, 1 uM, 3.3 uM, 10 uM, 33 uM, and 100uM). In one embodiment, the PL peptide is allowed to react with thebound GST/PDZ fusion protein (as well as the GST alone negative control)for 10 minutes at 4° C. followed by 20 minutes at 25° C. The plate iswashed 3 times with ice cold PBS to remove unbound labeled peptide.

(3) The binding of the PL peptide to the immobilized PDZ-domainpolypeptide is detected as described supra for the “G” assay.

(4) For each concentration of peptide, the net binding signal isdetermined by subtracting the binding of the peptide to GST alone fromthe binding of the peptide to the GST/PDZ fusion protein. The netbinding signal is then plotted as a function of ligand concentration andthe plot is fit (e.g. by using the Kaleidagraph software package curvefitting algorithm; Synergy Software) to the following equation, where“Signal_([ligand])” is the net binding signal at PL peptideconcentration “[ligand],” “Kd” is the apparent affinity of the bindingevent, and “Saturation Binding” is a constant determined by the curvefitting algorithm to optimize the fit to the experimental data:

Signal_([ligand])=Saturation Binding×([ligand]/([ligand]+Kd))

For reliable application of the above equation it is necessary that thehighest peptide ligand concentration successfully tested experimentallybe greater than, or at least similar to, the calculated Kd(equivalently, the maximum observed binding should be similar to thecalculated saturation binding). In cases where satisfying the abovecriteria proves difficult, an alternative approach (infra) can be used.

Approach 2:

(1) A fixed concentration of a PDZ-domain polypeptide and increasingconcentrations of a labeled PL peptide (labeled with, for example,biotin or fluorescein, see TABLE 3 for representative peptide amino acidsequences) are mixed together in solution and allowed to react. In oneembodiment, preferred peptide concentrations are 0.1 uM, 1 uM, 10 uM,100 uM, 1 mM. In various embodiments, appropriate reaction times canrange from 10 minutes to 2 days at temperatures ranging from 4° C. to37° C. In some embodiments, the identical reaction can also be carriedout using a non-PDZ domain-containing protein as a control (e.g., if thePDZ-domain polypeptide is fusion protein, the fusion partner can beused).

(2) PDZ-ligand complexes can be separated from unbound labeled peptideusing a variety of methods known in the art. For example, the complexescan be separated using high performance size-exclusion chromatography(HPSEC, gel filtration) (Rabinowitz et al., 1998, Immunity 9:699),affinity chromatography (e.g. using glutathione Sepharose beads), andaffinity absorption (e.g., by binding to an anti-GST-coated plate asdescribed supra).

(3) The PDZ-ligand complex is detected based on presence of the label onthe peptide ligand using a variety of methods and detectors known to oneof skill in the art. For example, if the label is fluorescein and theseparation is achieved using HPSEC, an in-line fluorescence detector canbe used. The binding can also be detected as described supra for the Gassay.

(4) The PDZ-ligand binding signal is plotted as a function of ligandconcentration and the plot is fit. (e.g., by using the Kaleidagraphsoftware package curve fitting algorithm) to the following equation,where “Signal_([ligand])” is the binding signal at PL peptideconcentration “[ligand],” “Kd” is the apparent affinity of the bindingevent, and “Saturation Binding” is a constant determined by the curvefitting algorithm to optimize the fit to the experimental data:

Signal_([Ligand])=Saturation Binding×([ligand]/([ligand+Kd])

Measurement of the affinity of a labeled peptide ligand binding to aPDZ-domain polypeptide is useful because knowledge of the affinity (orapparent affinity) of this interaction allows rational design ofinhibitors of the interaction with known potency. The potency ofinhibitors in inhibition would be similar to (i.e. within one-order ofmagnitude of) the apparent affinity of the labeled peptide ligandbinding to the PDZ-domain.

Thus, in one aspect, the invention provides a method of determining theapparent affinity of binding between a PDZ domain and a ligand byimmobilizing a polypeptide comprising the PDZ domain and a non-PDZdomain on a surface, contacting the immobilized polypeptide with aplurality of different concentrations of the ligand, determining theamount of binding of the ligand to the immobilized polypeptide at eachof the concentrations of ligand, and calculating the apparent affinityof the binding based on that data. Typically, the polypeptide comprisingthe PDZ domain and a non-PDZ domain is a fusion protein. In oneembodiment, the e.g., fusion protein is GST-PDZ fusion protein, butother polypeptides can also be used (e.g., a fusion protein including aPDZ domain and any of a variety of epitope tags, biotinylation signalsand the like) so long as the polypeptide can be immobilized In anorientation that does not abolish the ligand binding properties of thePDZ domain, e.g., by tethering the polypeptide to the surface via thenon-PDZ domain via an anti-domain antibody and leaving the PDZ domain asthe free end. It was discovered, for example, reacting a PDZ-GST fusionpolypeptide directly to a plastic plate provided suboptimal results. Thecalculation of binding affinity itself can be determined using anysuitable equation (e.g., as shown supra; also see Cantor and Schimmel(1980) BIOPHYSICAL CHEMISTRY WH Freeman & Co., San Francisco) orsoftware.

Thus, in a preferred embodiment, the polypeptide is immobilized bybinding the polypeptide to an immobilized immunoglobulin that binds thenon-PDZ domain (e.g., an anti-GST antibody when a GST-PDZ fusionpolypeptide is used). In a preferred embodiment, the step of contactingthe ligand and PDZ-domain polypeptide is carried out under theconditions provided supra in the description of the “G” assay. It willbe appreciated that binding assays are conveniently carried out inmultiwell plates (e.g., 24-well, 96-well plates, or 384 well plates).

The present method has considerable advantages over other methods formeasuring binding affinities PDZ-PL affinities, which typically involvecontacting varying concentrations of a GST-PDZ fusion protein to aligand-coated surface. For example, some previously described methodsfor determining affinity (e.g., using immobilized ligand and GST-PDZprotein in solution) did not account for oligomerization state of thefusion proteins used, resulting in potential errors of more than anorder of magnitude.

Although not sufficient for quantitative measurement of PDZ-PL bindingaffinity, an estimate of the relative strength of binding of differentPDZ-PL pairs can be made based on the absolute magnitude of the signalsobserved in the “G assay.” This estimate will reflect several factors,including biologically relevant aspects of the interaction, includingthe affinity and the dissociation rate. For comparisons of differentligands binding to a given PDZ domain-containing protein, differences inabsolute binding signal likely relate primarily to the affinity and/ordissociation rate of the interactions of interest.

IX. Measurements of Assay Specificity

As described supra, the present invention provides powerful methods foranalysis of PDZ-ligand interactions, including high-throughput methodssuch as the “G” assay and affinity assays described supra. In oneembodiment of the invention, the affinity is determined for a particularligand and a plurality of PDZ proteins. Typically the plurality is atleast 5, and often at least 25, or at least 40 different PDZ proteins.In a preferred embodiment, the plurality of different PDZ proteins arefrom a particular tissue (e.g., central nervous system, spleen, cardiacmuscle, kidney) or a particular class or type of cell, (e.g., ahematopoietic cell, a lymphocyte, a neuron) and the like. In a mostpreferred embodiment, the plurality of different PDZ proteins representsa substantial fraction (e.g., typically a majority, more often at least80%) of all of the PDZ proteins known to be, or suspected of being,expressed in the tissue or cell(s), e.g., all of the PDZ proteins knownto be present in lymphocytes. In an embodiment, the plurality is atleast 50%, usually at least 80%, at least 90% or all of the PDZ proteinsdisclosed herein as being expressed in hematopoietic cells.

In one embodiment of the invention, the binding of a ligand to theplurality of PDZ proteins is determined. Using this method, it ispossible to identify a particular PDZ domain bound with particularspecificity by the ligand. The binding may be designated as “specific”if the affinity of the ligand to the particular PDZ domain is at least2-fold that of the binding to other PDZ domains in the plurality (e.g.,present in that cell type). The binding is deemed “very specific” if theaffinity is at least 10-fold higher than to any other PDZ in theplurality or, alternatively, at least 10-fold higher than to at least90%, more often 95% of the other PDZs in a defined plurality. Similarly,the binding is deemed “exceedingly specific” if it is at least 100-foldhigher. For example, a ligand could bind to 2 different PDZs with anaffinity of 1 uM and to no other PDZs out of a set 40 with an affinityof less than 100 uM. This would constitute specific binding to those 2PDZs. Similar measures of specificity are used to describe binding of aPDZ to a plurality of PLs.

It will be recognized that high specificity PDZ-PL interactionsrepresent potentially more valuable targets for achieving a desiredbiological effect. The ability of an inhibitor or enhancer to act withhigh specificity is often desirable. In particular, the most specificPDZ-ligand interactions are also the diagnostic targets, allowingspecific detection of the interaction or disruption of an interaction.

Thus, in one embodiment, the invention provides a method of identifyinga high specificity interaction between a particular PDZ domain and aligand known or suspected of binding at least one PDZ domain, byproviding a plurality of different immobilized polypeptides, each ofsaid polypeptides comprising a PDZ domain and a non-PDZ domain;determining the affinity of the ligand for each of said polypeptides,and comparing the affinity of binding of the ligand to each of saidpolypeptides, wherein an interaction between the ligand and a particularPDZ domain is deemed to have high specificity when the ligand binds animmobilized polypeptide comprising the particular PDZ domain with atleast 2-fold higher affinity than to immobilized polypeptides notcomprising the particular PDZ domain.

In a related aspect, the affinity of binding of a specific PDZ domain toa plurality of ligands (or suspected ligands) is determined. Forexample, in one embodiment, the invention provides a method ofidentifying a high specificity interaction between a PDZ-domain and aparticular ligand known or suspected of binding at least one PDZ domain,by providing an immobilized polypeptide comprising the PDZ domain and anon-PDZ domain; determining the affinity of each of a plurality ofligands for the polypeptide, and comparing the affinity of binding ofeach of the ligands to the polypeptide, wherein an interaction between aparticular ligand and the PDZ domain is deemed to have high specificitywhen the ligand binds an immobilized polypeptide comprising the PDZdomain with at least 2-fold higher affinity than other ligands tested.Thus, the binding may be designated as “specific” if the affinity of thePDZ to the particular PL is at least 2-fold that of the binding to otherPLs in the plurality (e.g., present in that cell type). The binding isdeemed “very specific” if the affinity is at least 10-fold higher thanto any other PL in the plurality or, alternatively, at least 10-foldhigher than to at least 90%, more often 95% of the other PLs in adefined plurality. Similarly, the binding is deemed “exceedinglyspecific” if it is at least 100-fold higher. Typically the plurality isat least 5 different ligands, more often at least 10.

A. Use of Array for Global Predictions

One discovery of the present inventors relates to the important andextensive roles played by interactions between PDZ proteins and PLproteins, particularly in the biological function of hematopoietic cellsand other cells involved in the immune response. Further, it has beendiscovered that valuable information can be ascertained by analysis(e.g., simultaneous analysis) of a large number of PDZ-PL interactions.In a preferred embodiment, the analysis encompasses all of the PDZproteins expressed in a particular tissue (e.g., spleen) or type orclass of cell (e.g., hematopoietic cell, neuron, lymphocyte, B cell, Tcell and the like). Alternatively, the analysis encompasses at leastabout 5, or at least about 10, or at least about 12, or at least about15 and often at least 50 different polypeptides, up to about 60, about80, about 100, about 150, about 200, or even more differentpolypeptides; or a substantial fraction (e.g., typically a majority,more often at least 80%) of all of the PDZ proteins known to be, orsuspected of being, expressed in the tissue or cell(s), e.g., all of thePDZ proteins known to be present in lymphocytes.

It will be recognized that the arrays and methods of the invention aredirected to the analysis of PDZ and PL interactions, and involveselection of such proteins for analysis. While the devices and methodsof the invention may include or involve a small number of controlpolypeptides, they typically do not include significant numbers ofproteins or fusion proteins that do not include either PDZ or PL domains(e.g., typically, at least about 90% of the arrayed or immobilizedpolypeptides in a method or device of the invention is a PDZ or PLsequence protein, more often at least about 95%, or at least about 99%).

It will be apparent from this disclosure that analysis of the relativelylarge number of different interactions preferably takes placesimultaneously. In this context, “simultaneously” means that theanalysis of several different PDZ-PL interactions (or the effect of atest agent on such interactions) is assessed at the same time. Typicallythe analysis is carried out in a high throughput (e.g., robotic)fashion. One advantage of this method of simultaneous analysis is thatit permits rigorous comparison of multiple different PDZ-PLinteractions. For example, as explained in detail elsewhere herein,simultaneous analysis (and use of the arrays described infra)facilitates, for example, the direct comparison of the effect of anagent (e.g., an potential interaction inhibitor) on the interactionsbetween a substantial portion of PDZs and/or PLs in a tissue or cell.

Accordingly, in one aspect, the invention provides an array ofimmobilized polypeptide comprising the PDZ domain and a non-PDZ domainon a surface. Typically, the array comprises at least about 5, or atleast about 10, or at least about 12, or at least about 15 and often atleast 50 different polypeptides. In one preferred embodiment, thedifferent PDZ proteins are from a particular tissue (e.g., centralnervous system, spleen, cardiac muscle, kidney) or a particular class ortype of cell, (e.g., a hematopoietic cell, a lymphocyte, a neuron) andthe like. In a most preferred embodiment, the plurality of different PDZproteins represents a substantial fraction (e.g., typically a majority,more often at least 60%, 70% or 80%) of all of the PDZ proteins known tobe, or suspected of being, expressed in the tissue or cell(s), e.g., allof the PDZ proteins known to be present in lymphocytes.

Certain embodiments are arrays which include a plurality, usually atleast 5, 10, 25, 50 PDZ proteins present in a particular cell ofinterest. In this context, “array” refers to an ordered series ofimmobilized polypeptides in which the identity of each polypeptide isassociated with its location. In some embodiments the plurality ofpolypeptides are arrayed in a “common” area such that they can besimultaneously exposed to a solution (e.g., containing a ligand or testagent). For example, the plurality of polypeptides can be on a slide,plate or similar surface, which may be plastic, glass, metal, silica,beads or other surface to which proteins can be immobilized. In adifferent embodiment, the different immobilized polypeptides aresituated in separate areas, such as different wells of multi-well plate(e.g., a 24-well plate, a 96-well plate, a 384 well plate, and thelike). It will be recognized that a similar advantage can be obtained byusing multiple arrays in tandem.

B. Analysis of PDZ-PL Inhibition Profile

In one aspect, the invention provides a method for determining if a testcompound inhibits any PDZ-ligand interaction in large set of PDZ-ligandinteractions (e.g., a plurality of the PDZ-ligands interactionsdescribed in U.S. patent application Ser. No. 09/724,553; a majority ofthe PDZ-ligands identified in a particular cell or tissue as describedsupra (e.g., cervical tissue) and the like. In one embodiment, the PDZdomains of interest are expressed as GST-PDZ fusion proteins andimmobilized as described herein. For each PDZ domain, a labeled ligandthat binds to the domain with a known affinity is identified asdescribed herein.

For any known or suspected modulator (e.g., inhibitor) of a PDZ-PLinteraction(s), it is useful to know which interactions are inhibited(or augmented). This information could be used as a diagnostic markerfor the presence of a pathogen (e.g., oncogenic HPV strains). Theprofile of PDZ interactions inhibited by a particular agent is referredto as the “inhibition profile” for the agent, and is described in detailbelow. The profile of PDZ interactions enhanced by a particular agent isreferred to as the “enhancement profile” for the agent. It will bereadily apparent to one of skill guided by the description of theinhibition profile how to determine the enhancement profile for anagent. The present invention provides methods for determining the PDZinteraction (inhibition/enhancement) profile of an agent in a singleassay.

In one aspect, the invention provides a method for determining thePDZ-PL inhibition profile of a compound by providing (i) a plurality ofdifferent immobilized polypeptides, each of said polypeptides comprisinga PDZ domain and a non-PDZ domain and (ii) a plurality of correspondingligands, wherein each ligand binds at least one PDZ domain in (i), thencontacting each of said immobilized polypeptides in (i) with acorresponding ligand in (ii) in the presence and absence of a testcompound, and determining for each polypeptide-ligand pair whether thetest compound inhibits binding between the immobilized polypeptide andthe corresponding ligand.

Typically the plurality is at least 5, and often at least 25, or atleast 40 different PDZ proteins. In a preferred embodiment, theplurality of different ligands and the plurality of different PDZproteins are from the same tissue or a particular class or type of cell,e.g., a cervical cell, a penile cell, an anal cell and the like. In amost preferred embodiment, the plurality of different PDZs represents asubstantial fraction (e.g., at least 80%) of all of the PDZs known tobe, or suspected of being, expressed in the tissue or cell(s), e.g., allof the PDZs known to be present in lymphocytes (for example, at least80%, at least 90% or all of the PDZs disclosed herein as being expressedin hematopoietic cells).

In one embodiment, the inhibition profile is determined as follows: Aplurality (e.g., all known) PDZ domains expressed in a cell (e.g.,cervical cells) are expressed as GST-fusion proteins and immobilizedwithout altering their ligand binding properties as described supra. Foreach PDZ domain, a labeled ligand that binds to this domain with a knownaffinity is identified. If the set of PDZ domains expressed inlymphocytes is denoted by {P1 . . . Pn}, any given PDZ domain Pi binds a(labeled) ligand Li with affinity K_(d)i. To determine the inhibitionprofile for a test agent “compound X” the “G” assay (supra) can beperformed as follows in 96-well plates with rows A-H and columns 1-12.Column 1 is coated with P1 and washed. The corresponding ligand L1 isadded to each washed coated well of column I at a concentration 0.5K_(d)1 with (rows B, D, F, H) or without (rows A, C, E, F) between about1 and about 1000 uM) of test compound X. Column 2 is coated with P2, andL2 (at a concentration 0.5 K_(d)2) is added with or without inhibitor X.Additional PDZ domains and ligands are similarly tested.

Compound X is considered to inhibit the binding of Li to Pi if theaverage signal in the wells of column i containing X is less than halfthe signal in the equivalent wells of the column lacking X. Thus, inthis single assay one determines the full set of lymphocyte PDZs thatare inhibited by compound X.

In some embodiments, the test compound X is a mixture of compounds, suchas the product of a combinatorial chemistry synthesis as describedsupra. In some embodiments, the test compound is known to have a desiredbiological effect, and the assay is used to determine the mechanism ofaction (i.e., if the biological effect is due to modulating a PDZ-PLinteraction).

It will be apparent that an agent that modulates only one, or a fewPDZ-PL interactions, in a panel (e.g., a panel of all known PDZslymphocytes, a panel of at least 10, at least 20 or at least 50 PDZdomains) is a more specific modulator than an agent that modulate manyor most interactions. Typically, an agent that modulates less than 20%of PDZ domains in a panel (e.g., Table 2) is deemed a “specific”inhibitor, less than 6% a “very specific” inhibitor, and a single PDZdomain a “maximally specific” inhibitor.

It will also be appreciated that “compound X” may be a compositioncontaining mixture of compounds (e.g., generated using combinatorialchemistry methods) rather than a single compound.

Several variations of this assay are contemplated:

In some alternative embodiments, the assay above is performed usingvarying concentrations of the test compound X, rather than fixedconcentration. This allows determination of the Ki of the X for each PDZas described above.

In an alternative embodiment, instead of pairing each PDZ-PL with aspecific labeled ligand Li, a mixture of different labeled ligands iscreated that such that for every PDZ at least one of the ligands in themixture binds to this PDZ sufficiently to detect the binding in the “G”assay. This mixture is then used for every PDZ domain.

In one embodiment, compound X is known to have a desired biologicaleffect, but the chemical mechanism by which it has that effect isunknown. The assays of the invention can then be used to determine ifcompound X has its effect by binding to a PDZ domain.

In one embodiment, PDZ-domain containing proteins are classified in togroups based on their biological function, e.g. into those that regulatechemotaxis versus those that regulate transcription. An optimalinhibitor of a particular function (e.g., including but not limited toan anti-chemotactic agent, an anti-T cell activation agent, cell-cyclecontrol, vesicle transport, apoptosis, etc.) will inhibit multiplePDZ-ligand interactions involved in the function (e.g., chemotaxis,activation) but few other interactions. Thus, the assay is used in oneembodiment in screening and design of a drug that specifically blocks aparticular function. For example, an agent designed to block chemotaxismight be identified because, at a given concentration, the agentinhibits 2 or more PDZs involved in chemotaxis but fewer than 3 otherPDZs, or that inhibits PDZs involved in chemotaxis with a Ki>10-foldbetter than for other PDZs. Thus, the invention provides a method foridentifying an agent that inhibits a first selected PDZ-PL interactionor plurality of interactions but does not inhibit a second selectedPDZ-PL interaction or plurality of interactions. The two (or more) setsof interactions can be selected on the basis of the known biologicalfunction of the PDZ proteins, the tissue specificity of the PDZproteins, or any other criteria. Moreover, the assay can be used todetermine effective doses (i.e., drug concentrations) that result indesired biological effects while avoiding undesirable effects.

C. Agonists and Antagonists of PDZ-PL Interactions

As described herein, interactions between PDZ proteins and PL proteinsin cells (e.g., cervical cells) may be disrupted or inhibited by thepresence of pathogens. Pathogens can be identified using screeningassays described herein. Agonists and antagonists of PDZ-Pathogen PLinteractions or PDZ-Cellular PL interactions can be useful in discerningor confirming specific interactions. In some embodiments, an agonistwill increase the sensitivity of a PDZ-pathogen PL interaction. In otherembodiments, an antagonist of a PDZ-pathogen PL interaction can be usedto verify the specificity of an interaction. In one embodiment, themotifs disclosed herein are used to design inhibitors. In someembodiments, the antagonists of the invention have a structure (e.g.,peptide sequence) based on the C-terminal residues of PL-domain proteinslisted in TABLE 3. In some embodiments, the antagonists of the inventionhave a structure (e.g., peptide sequence) based on a PL motif disclosedherein or in U.S. patent application Ser. No. 09/724,553.

The PDZ/PL antagonists and antagonists of the invention may be any of alarge variety of compounds, both naturally occurring and synthetic,organic and inorganic, and including polymers (e.g., oligopeptides,polypeptides, oligonucleotides, and polynucleotides), small molecules,antibodies, sugars, fatty acids, nucleotides and nucleotide analogs,analogs of naturally occurring structures (e.g., peptide mimetics,nucleic acid analogs, and the like), and numerous other compounds.Although, for convenience, the present discussion primarily refersantagonists of PDZ-PL interactions, it will be recognized that PDZ-PLinteraction agonists can also be use in the methods disclosed herein.

In one aspect, the peptides and peptide mimetics or analogues of theinvention contain an amino acid sequence that binds a PDZ domain in acell of interest. In one embodiment, the antagonists comprise a peptidethat has a sequence corresponding to the carboxy-terminal sequence of aPL protein listed in TABLE 3 or in U.S. patent application Ser. No.09/724,553, e.g., a peptide listed TABLE 3. Typically, the peptidecomprises at least the C-terminal two (3), three (3) or four (4)residues of the PL protein, and often the inhibitory peptide comprisesmore than four residues (e.g., at least five, six, seven, eight, nine,ten, twelve or fifteen residues) from the PL protein C-terminus.

In some embodiments, the inhibitor is a peptide, e.g., having a sequenceof a PL C-terminal protein sequence.

In some embodiments, the antagonist is a fusion protein comprising sucha sequence. Fusion proteins containing a transmembrane transporter aminoacid sequence are particularly useful.

In some embodiments, the inhibitor is conserved variant of the PLC-terminal protein sequence having inhibitory activity.

In some embodiments, the antagonist is a peptide mimetic of a PLC-terminal sequence.

In some embodiments, the inhibitor is a small molecule (i.e., having amolecular weight less than 1 kD).

D. Peptide Antagonists

In one embodiment, the antagonists comprise a peptide that has asequence of a PL protein carboxy-terminus listed in TABLE 3. The peptidecomprises at least the C-terminal two (2) residues of the PL protein,and typically, the inhibitory peptide comprises more than two residues(e.g., at least three, four, five, six, seven, eight, nine, ten, twelveor fifteen residues) from the PL protein C-terminus. The peptide may beany of a variety of lengths (e.g., at least 2, at least 3, at least 4,at least 5, at least 6, at least 8, at least 10, or at least 20residues) and may contain additional residues not from the PL protein.It will be recognized that short PL peptides are sometime used in therational design of other small molecules with similar properties.

Although most often, the residues shared by the inhibitory peptide withthe PL protein are found at the C-terminus of the peptide. However, insome embodiments, the sequence is internal. Similarly, in some cases,the inhibitory peptide comprises residues from a PL sequence that isnear, but not at the c-terminus of a PL protein (see, Gee et al., 1998,J. Biological Chem. 273:21980-87).

Sometime the PL protein carboxy-terminus sequence is referred to as the“core PDZ motif sequence” referring to the ability of the short sequenceto interact with the PDZ domain. For example, in an embodiment, the“core PDZ motif sequence” contains the last four C-terminus amino acids.As described above, the four amino acid core of a PDZ motif sequence maycontain additional amino acids at its amino terminus to further increaseits binding affinity and/or stability. Thus, in one embodiment, the PDZmotif sequence peptide can be from four amino acids up to 15 aminoacids. It is preferred that the length of the sequence to be 6-10 aminoacids. More preferably, the PDZ motif sequence contains 8 amino acids.Additional amino acids at the amino terminal end of the core sequencemay be derived from the natural sequence in each hematopoietic cellsurface receptor or a synthetic linker. The additional amino acids mayalso be conservatively substituted. When the third residue from theC-terminus is S, T or Y, this residue may be phosphorylated prior to theuse of the peptide.

In some embodiments, the peptide and nonpeptide inhibitors of the aresmall, e.g., fewer than ten amino acid residues in length if a peptide.Further, it is reported that a limited number of ligand amino acidsdirectly contact the PDZ domain (generally less than eight) (Kozlov etal., 2000, Biochemistry 39, 2572; Doyle et al., 1996, Cell 85, 1067) andthat peptides as short as the C-terminal three amino acids often retainsimilar binding properties to longer (>15) amino acids peptides(Yanagisawa et al., 1997, J. Biol. Chem. 272, 8539).

E. Peptide Variants

Having identified PDZ binding peptides and PDZ-PL interaction inhibitorysequences, variations of these sequences can be made and the resultingpeptide variants can be tested for PDZ domain binding or PDZ-PLinhibitory activity. In embodiments, the variants have the same or adifferent ability to bind a PDZ domain as the parent peptide. Typically,such amino acid substitutions are conservative, i.e., the amino acidresidues are replaced with other amino acid residues having physicaland/or chemical properties similar to the residues they are replacing.Preferably, conservative amino acid substitutions are those wherein anamino acid is replaced with another amino acid encompassed within thesame designated class.

F. Peptide Mimetics

Having identified PDZ binding peptides and PDZ-PL interaction inhibitorysequences, peptide mimetics can be prepared using routine methods, andthe inhibitory activity of the mimetics can be confirmed using theassays of the invention. Thus, in some embodiments, the agonist orantagonist is a peptide mimetic of a PL C-terminal sequence. The skilledartisan will recognize that individual synthetic residues andpolypeptides incorporating mimetics can be synthesized using a varietyof procedures and methodologies, which are well described in thescientific and patent literature, e.g., Organic Syntheses CollectiveVolumes, Gilman et al. (Eds) John Wiley & Sons, Inc., NY. Polypeptidesincorporating mimetics can also be made using solid phase syntheticprocedures, as described, e.g., by Di Marchi, et al., U.S. Pat. No.5,422,426. Mimetics of the invention can also be synthesized usingcombinatorial methodologies. Various techniques for generation ofpeptide and peptidomimetic libraries are well known, and include, e.g.,multipin, tea bag, and split-couple-mix techniques; see, e.g., al-Obeidi(1998) Mol. Biotechnol. 9:205-223; Hruby (1997) Curr. Opin. Chem. Biol.1:114-119; Ostergaard (1997) Mol. Divers. 3:17-27; Ostresh (1996)Methods Enzymol. 267:220-234.

G. Small Molecules

In some embodiments, the agonist or antagonist is a small molecule(i.e., having a molecular weight less than 1 kD). Methods for screeningsmall molecules are well known in the art and include those describedsupra.

X. Methods of Optimizing a PL Detector

Although described supra primarily in terms of identifying interactionsbetween PDZ-domain polypeptides and PL proteins, the assays describedsupra and other assays can also be used to identify the binding of othermolecules (e.g., peptide mimetics, small molecules, and the like) to PDZdomain sequences. For example, using the assays disclosed herein,combinatorial and other libraries of compounds can be screened, e.g.,for molecules that specifically bind to PDZ domains. Screening oflibraries can be accomplished by any of a variety of commonly knownmethods. See, e.g., the following references, which disclose screeningof peptide libraries: Parmley and Smith, 1989, Adv. Exp. Med. Biol.251:215-218; Scott and Smith, 1990, Science 249:386-390; Fowlkes et al.,1992; BioTechniques 13:422-427; Oldenburg et al., 1992, Proc. Natl.Acad. Sci. USA 89:5393-5397; Yu et al., 1994, Cell 76:933-945; Staudt etal., 1988, Science 241:577-580; Bock et al., 1992, Nature 355:564-566;Tuerk et al., 1992, Proc. Natl. Acad. Sci. USA 89:6988-6992; Ellingtonet al., 1992, Nature 355:850-852; U.S. Pat. No. 5,096,815, U.S. Pat. No.5,223,409, and U.S. Pat. No. 5,198,346, all to Ladner et al.; Rebar andPabo, 1993, Science 263:671-673; and PCT Publication No. WO 94/18318.

In a specific embodiment, screening can be carried out by contacting thelibrary members with a PDZ-domain polypeptide immobilized on a solidsupport (e.g. as described supra in the “G” assay) and harvesting thoselibrary members that bind to the protein. Examples of such screeningmethods, termed “panning” techniques are described by way of example inParmley and Smith, 1988, Gene 73:305-318; Fowlkes et al., 1992,BioTechniques 13:422-427; PCT Publication No. WO 94/18318; and inreferences cited hereinabove.

In another embodiment, the two-hybrid system for selecting interactingproteins in yeast (Fields and Song, 1989, Nature 340:245-246; Chien etal., 1991, Proc. Natl. Acad. Sci. USA 88:9578-9582) can be used toidentify molecules that specifically bind to a PDZ domain-containingprotein. Furthermore, the identified molecules are further tested fortheir ability to inhibit transmembrane receptor interactions with a PDZdomain.

In one aspect of the invention, antagonists of an interaction between aPDZ protein and a PL protein are identified. In one embodiment, amodification of the “A” assay described supra is used to identifyantagonists. In one embodiment, a modification of the “G” assaydescribed supra is used to identify antagonists.

In one embodiment, screening assays are used to detect molecules thatspecifically bind to PDZ domains. Such molecules are useful as agonistsor antagonists of PDZ-protein-mediated cell function (e.g., cellactivation, e.g., T cell activation, vesicle transport, cytokinerelease, growth factors, transcriptional changes, cytoskeletonrearrangement, cell movement, chemotaxis, and the like). In oneembodiment, such assays are performed to screen for leukocyte activationinhibitors for drug development. The invention thus provides assays todetect molecules that specifically bind to PDZ domain-containingproteins. For example, recombinant cells expressing PDZ domain-encodingnucleic acids can be used to produce PDZ domains in these assays and toscreen for molecules that bind to the domains. Molecules are contactedwith the PDZ domain (or fragment thereof) under conditions conducive tobinding, and then molecules that specifically bind to such domains areidentified. Methods that can be used to carry out the foregoing arecommonly known in the art.

It will be appreciated by the ordinarily skilled practitioner that, inone embodiment, antagonists are identified by conducting the A or Gassays in the presence and absence of a known or candidate antagonist.When decreased binding is observed in the presence of a compound, thatcompound is identified as an antagonist. Increased binding in thepresence of a compound signifies that the compound is an agonist.

For example, in one assay, a test compound can be identified as aninhibitor (antagonist) of binding between a PDZ protein and a PL proteinby contacting a PDZ domain polypeptide and a PL peptide in the presenceand absence of the test compound, under conditions in which they would(but for the presence of the test compound) form a complex, anddetecting the formation of the complex in the presence and absence ofthe test compound. It will be appreciated that less complex formation inthe presence of the test compound than in the absence of the compoundindicates that the test compound is an inhibitor of a PDZ protein-PLprotein binding.

In one embodiment, the “G” assay is used in the presence or absence of acandidate inhibitor. In one embodiment, the “A” assay is used in thepresence or absence of a candidate inhibitor.

In one embodiment (in which a G assay is used), one or more PDZdomain-containing GST-fusion proteins are bound to the surface of wellsof a 96-well plate as described supra (with appropriate controlsincluding nonfusion GST protein). All fusion proteins are bound inmultiple wells so that appropriate controls and statistical analysis canbe done. A test compound in BSA/PBS (typically at multiple differentconcentrations) is added to wells. Immediately thereafter, 30 uL of adetectably labeled (e.g., biotinylated) peptide known to bind to therelevant PDZ domain (see, e.g., TABLE 4) is added in each of the wellsat a final concentration of, e.g., between about 2 uM and about 40 uM,typically 5 uM, 15 uM, or 25 uM. This mixture is then allowed to reactwith the PDZ fusion protein bound to the surface for 10 minutes at 4° C.followed by 20 minutes at 25° C. The surface is washed free of unboundpeptide three times with ice cold PBS and the amount of binding of thepeptide in the presence and absence of the test compound is determined.Usually, the level of binding is measured for each set of replica wells(e.g. duplicates) by subtracting the mean GST alone background from themean of the raw measurement of peptide binding in these wells.

In an alternative embodiment, the A assay is carried out in the presenceor absence of a test candidate to identify inhibitors of PL-PDZinteractions.

In one embodiment, a test compound is determined to be a specificinhibitor of the binding of the PDZ domain (P) and a PL (L) sequencewhen, at a test compound concentration of less than or equal to 1 mM(e.g., less than or equal to: 500 uM, 100 uM, 10 uM, 1 uM, 100 nM or 1nM) the binding of P to L in the presence of the test compound less thanabout 50% of the binding in the absence of the test compound. (invarious embodiments, less than about 25%, less than about 10%, or lessthan about 1%). Preferably, the net signal of binding of P to L in thepresence of the test compound plus six (6) times the standard error ofthe signal in the presence of the test compound is less than the bindingsignal in the absence of the test compound.

In one embodiment, assays for an inhibitor are carried out using asingle PDZ protein-PL protein pair (e.g., a PDZ domain fusion proteinand a PL peptide). In a related embodiment, the assays are carried outusing a plurality of pairs, such as a plurality of different pairslisted in TABLE 4.

In some embodiments, it is desirable to identify compounds that, at agiven concentration, inhibit the binding of one PL-PDZ pair, but do notinhibit (or inhibit to a lesser degree) the binding of a specifiedsecond PL-PDZ pair. These antagonists can be identified by carrying outa series of assays using a candidate inhibitor and different PL-PDZpairs (e.g., as shown in the matrix of TABLE 4) and comparing theresults of the assays. All such pairwise combinations are contemplatedby the invention (e.g., test compound inhibits binding of PL₁ to PDZ₁ toa greater degree than it inhibits binding of PL₁ to PDZ₂ or PL₂ toPDZ₂). Importantly, it will be appreciated that, based on the dataprovided in TABLE 4 and disclosed herein (and additional data that canbe generated using the methods described herein) inhibitors withdifferent specificities can readily be designed.

For example, according to the invention, the Ki (“potency”) of aninhibitor of a PDZ-PL interaction can be determined. Ki is a measure ofthe concentration of an inhibitor required to have a biological effect.For example, administration of an inhibitor of a PDZ-PL interaction inan amount sufficient to result in an intracellular inhibitorconcentration of at least between about 1 and about 100 Ki is expectedto inhibit the biological response mediated by the target PDZ-PLinteraction. In one aspect of the invention, the Kd measurement ofPDZ-PL binding as determined using the methods supra is used indetermining Ki.

Thus, in one aspect, the invention provides a method of determining thepotency (Ki) of an inhibitor or suspected inhibitor of binding between aPDZ domain and a ligand by immobilizing a polypeptide comprising the PDZdomain and a non-PDZ domain on a surface, contacting the immobilizedpolypeptide with a plurality of different mixtures of the ligand andinhibitor, wherein the different mixtures comprise a fixed amount ofligand and different concentrations of the inhibitor, determining theamount of ligand bound at the different concentrations of inhibitor, andcalculating the Ki of the binding based on the amount of ligand bound inthe presence of different concentrations of the inhibitor. In anembodiment, the polypeptide is immobilized by binding the polypeptide toan immobilized immunoglobulin that binds the non-PDZ domain. Thismethod, which is based on the “G” assay described supra, is particularlysuited for high-throughput analysis of the Ki for inhibitors ofPDZ-ligand interactions. Further, using this method, the inhibition ofthe PDZ-ligand interaction itself is measured, without distortion ofmeasurements by avidity effects.

Typically, at least a portion of the ligand is detectably labeled topermit easy quantitation of ligand binding.

It will be appreciated that the concentration of ligand andconcentrations of inhibitor are selected to allow meaningful detectionof inhibition. Thus, the concentration of the ligand whose binding is tobe blocked is close to or less than its binding affinity (e.g.,preferably less than the 5×Kd of the interaction, more preferably lessthan 2×Kd, most preferably less than 1×Kd). Thus, the ligand istypically present at a concentration of less than 2 Kd (e.g., betweenabout 0.01 Kd and about 2 Kd) and the concentrations of the testinhibitor typically range from 1 nM to 100 uM (e.g. a 4-fold dilutionseries with highest concentration 10 uM or 1 mM). In a preferredembodiment, the Kd is determined using the assay disclosed supra.

The Ki of the binding can be calculated by any of a variety of methodsroutinely used in the art, based on the amount of ligand bound in thepresence of different concentrations of the inhibitor. In anillustrative embodiment, for example, a plot of labeled ligand bindingversus inhibitor concentration is fit to the equation:

S _(inhibitor) =S ₀ *Ki/([I]+Ki)

where S_(inhibitor) is the signal of labeled ligand binding toimmobilized PDZ domain in the presence of inhibitor at concentration [I]and S₀ is the signal in the absence of inhibitor (i.e., [I]=0).Typically [I] is expressed as a molar concentration.

In another aspect of the invention, an enhancer (sometimes referred toas, augmentor or agonist) of binding between a PDZ domain and a ligandis identified by immobilizing a polypeptide comprising the PDZ domainand a non-PDZ domain on a surface, contacting the immobilizedpolypeptide with the ligand in the presence of a test agent anddetermining the amount of ligand bound, and comparing the amount ofligand bound in the presence of the test agent with the amount of ligandbound by the polypeptide in the absence of the test agent. At leasttwo-fold (often at least 5-fold) greater binding in the presence of thetest agent compared to the absence of the test agent indicates that thetest agent is an agent that enhances the binding of the PDZ domain tothe ligand. As noted supra, agents that enhance PDZ-ligand interactionsare useful for disruption (dysregulation) of biological events requiringnormal PDZ-ligand function (e.g., cancer cell division and metastasis,and activation and migration of immune cells).

The invention also provides methods for determining the “potency” or“K_(enhancer)” of an enhancer of a PDZ-ligand interaction. For example,according to the invention, the K_(enhancer) of an enhancer of a PDZ-PLinteraction can be determined, e.g., using the Kd of PDZ-PL binding asdetermined using the methods described supra. K_(enhancer) is a measureof the concentration of an enhancer expected to have a biologicaleffect. For example, administration of an enhancer of a PDZ-PLinteraction in an amount sufficient to result in an intracellularinhibitor concentration of at least between about 0.1 and about 100K_(enhancer) (e.g., between about 0.5 and about 50 K_(enhancer)) isexpected to disrupt the biological response mediated by the targetPDZ-PL interaction.

Thus, in one aspect the invention provides a method of determining thepotency (K_(enhancer)) of an enhancer or suspected enhancer of bindingbetween a PDZ domain and a ligand by immobilizing a polypeptidecomprising the PDZ domain and a non-PDZ domain on a surface, contactingthe immobilized polypeptide with a plurality of different mixtures ofthe ligand and enhancer, wherein the different mixtures comprise a fixedamount of ligand, at least a portion of which is detectably labeled, anddifferent concentrations of the enhancer, determining the amount ofligand bound at the different concentrations of enhancer, andcalculating the potency (K_(enhancer)) of the enhancer from the bindingbased on the amount of ligand bound in the presence of differentconcentrations of the enhancer. Typically, at least a portion of theligand is detectably labeled to permit easy quantitation of ligandbinding. This method, which is based on the “G” assay described supra,is particularly suited for high-throughput analysis of the K_(enhancer)for enhancers of PDZ-ligand interactions.

It will be appreciated that the concentration of ligand andconcentrations of enhancer are selected to allow meaningful detection ofenhanced binding. Thus, the ligand is typically present at aconcentration of between about 0.01 Kd and about 0.5 Kd and theconcentrations of the test agent/enhancer typically range from 1 nM to 1mM (e.g. a 4-fold dilution series with highest concentration 10 uM or 1mM). In a preferred embodiment, the Kd is determined using the assaydisclosed supra.

The potency of the binding can be determined by a variety of standardmethods based on the amount of ligand bound in the presence of differentconcentrations of the enhancer or augmentor. For example, a plot oflabeled ligand binding versus enhancer concentration can be fit to theequation:

S([E])=S(0)+(S(0)*(D _(enhancer)−1)*[E]/([E]+K _(enhancer))

where “K_(enhancer)” is the potency of the augmenting compound, and“D_(enhancer)” is the fold-increase in binding of the labeled ligandobtained with addition of saturating amounts of the enhancing compound,[E] is the concentration of the enhancer. It will be understood thatsaturating amounts are the amount of enhancer such that further additiondoes not significantly increase the binding signal. Knowledge of“K_(enhancer)” is useful because it describes a concentration of theaugmenting compound in a target cell that will result in a biologicaleffect due to dysregulation of the PDZ-PL interaction. Typicaltherapeutic concentrations are between about 0.1 and about 100K_(enhancer).

For certain of the PDZ proteins and PL proteins shown to bind togetherand for which Kd values had been obtained, additional testing wasconducted to determine whether certain pharmaceutical compounds wouldact to antagonize or agonize the interactions. Assays were conducted asfor the G′ assay described supra both in the presence and absence oftest compound, except that 50 ul of a 10 uM solution of the biotinylatedPL peptide is allowed to react with the surface bearing the PDZ-domainpolypeptide instead of a 20 uM solution as specified in step (2) of theassay.

Another method of increasing the specificity or sensitivity of a PDZ-PLinteraction is through mutagenesis and selection of high affinity orhigh specificity variants. Methods such as V, chemical (e.g., EMS) orbiological mutagenesis (e.g. Molecular shuffling or DNA polymerasemutagenesis) can be applied to create mutations in DNA encoding PDZdomains or PL domains. Proteins can then be made from variants andtested using a number of methods described herein (e.g., ‘A’ assay, ‘G’assay or yeast two hybrid). In general, one would assay mutants for highaffinity binding between the mutated PDZ domain and a test sample (suchas an oncogenic E6 PL) that have reduced affinity for other cellular PLs(as described in section IX). These methods are known to those skilledin the art and examples herein are not intended to be limiting.

XI. Recombinant Detector Synthesis

As indicated in the Background section, PDZ domain-containing proteinsare involved in a number of biological functions, including, but notlimited to, vesicular trafficking, tumor suppression, protein sorting,establishment of membrane polarity, apoptosis, regulation of immuneresponse and organization of synapse formation. In general, this familyof proteins has a common function of facilitating the assembly ofmulti-protein complexes, often serving as a bridge between severalproteins, or regulating the function of other proteins. Additionally, asalso noted supra, these proteins are found in essentially all celltypes. Consequently, detection of inappropriate PDZ:PL interactions orabnormal interactions can be utilized to diagnose a wide variety ofbiological and physiological conditions. In particular, detection of PLproteins from pathogenic organisms can be diagnosed using PDZ domains.Most, but not all, embodiments of this invention, require the additionof a detectable marker to the PDZ or PL protein used for detection.Examples are given below.

A. Chemical Synthesis

The peptides of the invention or analogues thereof, may be preparedusing virtually any art-known technique for the preparation of peptidesand peptide analogues. For example, the peptides may be prepared inlinear form using conventional solution or solid phase peptide synthesesand cleaved from the resin followed by purification procedures(Creighton, 1983, Protein Structures And Molecular Principles, W.H.Freeman and Co., N.Y.). Suitable procedures for synthesizing thepeptides described herein are well known in the art. The composition ofthe synthetic peptides may be confirmed by amino acid analysis orsequencing (e.g., the Edman degradation procedure and massspectroscopy).

In addition, analogues and derivatives of the peptides can be chemicallysynthesized. The linkage between each amino acid of the peptides of theinvention may be an amide, a substituted amide or an isostere of amide.Nonclassical amino acids or chemical amino acid analogues can beintroduced as a substitution or addition into the sequence.Non-classical amino acids include, but are not limited to, the D-isomersof the common amino acids, α-amino isobutyric acid, 4-aminobutyric acid,Abu, 2-amino butyric acid, γ-Abu, ε-Ahx, 6-amino hexanoic acid, Aib,2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine,norvaline, hydroxyproline, sarcosine, citrulline, cysteic acid,t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine,β-alanine, fluoro-amino acids, designer amino acids such as β-methylamino acids, C_(α)-methyl amino acids, N_(α)-methyl amino acids, andamino acid analogues in general. Furthermore, the amino acid can be D(dextrorotary) or L (levorotary).

B. Recombinant Synthesis

If the peptide is composed entirely of gene-encoded amino acids, or aportion of it is so composed, the peptide or the relevant portion mayalso be synthesized using conventional recombinant genetic engineeringtechniques. For recombinant production, a polynucleotide sequenceencoding a linear form of the peptide is inserted into an appropriateexpression vehicle, i.e., a vector which contains the necessary elementsfor the transcription and translation of the inserted coding sequence,or in the case of an RNA viral vector, the necessary elements forreplication and translation. The expression vehicle is then transfectedinto a suitable target cell which will express the peptide. Depending onthe expression system used, the expressed peptide is then isolated byprocedures well-established in the art. Methods for recombinant proteinand peptide production are well known in the art (see, e.g., Maniatis etal., 1989, Molecular Cloning A Laboratory Manual, Cold Spring HarborLaboratory, N.Y.; and Ausubel et al., 1989, Current Protocols inMolecular Biology, Greene Publishing Associates and Wiley Interscience,N.Y.).

A variety of host-expression vector systems may be utilized to expressthe peptides described herein. These include, but are not limited to,microorganisms such as bacteria transformed with recombinantbacteriophage DNA or plasmid DNA expression vectors containing anappropriate coding sequence; yeast or filamentous fungi transformed withrecombinant yeast or fungi expression vectors containing an appropriatecoding sequence; insect cell systems infected with recombinant virusexpression vectors (e.g., baculovirus) containing an appropriate codingsequence; plant cell systems infected with recombinant virus expressionvectors (e.g., cauliflower mosaic virus or tobacco mosaic virus) ortransformed with recombinant plasmid expression vectors (e.g., Tiplasmid) containing an appropriate coding sequence; or animal cellsystems.

In some embodiments, increasing the number of copies of a PL detectormay be used to increase the specificity or sensitivity of detection. Anexample of this is presented in EXAMPLE 4. The TIP-TIP-IgG vectorproduces a fusion protein that has duplicated copies of the PDZ domainfrom TIP-1 and the protein itself should dimerize on the basis of theIgG constant region backbone. Hence, a single protein contains 2-4copies of the TIP-1 PDZ domain. In a similar manner, addition tandemrepeats of PL detectors could be fashioned. In some embodiments,different PDZ domains from different proteins could be engineered toexpress as a single protein (e.g., the PDZ domains of TIP-1 and MAGI-1could be engineered to detect oncogenic HPV E6 proteins). In a similarmanner, a different Ig backbone could be used to increase the avidity ofa construct. For example, the IgG constant regions will dimerize withitself, but the IgM constant regions will form a complex of tenmonomers.

The expression elements of the expression systems vary in their strengthand specificities. Depending on the host/vector system utilized, any ofa number of suitable transcription and translation elements, includingconstitutive and inducible promoters, may be used in the expressionvector. For example, when cloning in bacterial systems, induciblepromoters such as pL of bacteriophage λ, plac, ptrp, ptac (ptrp-lachybrid promoter) and the like may be used; when cloning in insect cellsystems, promoters such as the baculovirus polyhedron promoter may beused; when cloning in plant cell systems, promoters derived from thegenome of plant cells (e.g., heat shock promoters; the promoter for thesmall subunit of RUBISCO; the promoter for the chlorophyll afb bindingprotein) or from plant viruses (e.g., the 35S RNA promoter of CaMV; thecoat protein promoter of TMV) may be used; when cloning in mammaliancell systems, promoters derived from the genome of mammalian cells(e.g., metallothionein promoter) or from mammalian viruses (e.g., theadenovirus late promoter; the vaccinia virus 7.5 K promoter) may beused; when generating cell lines that contain multiple copies ofexpression product, SV40-, BPV- and EBV-based vectors may be used withan appropriate selectable marker.

In cases where plant expression vectors are used, the expression ofsequences encoding the peptides of the invention may be driven by any ofa number of promoters. For example, viral promoters such as the 35S RNAand 19S RNA promoters of CaMV (Brisson et al., 1984, Nature310:511-514), or the coat protein pro moter of TMV (Takamatsu et al.,1987, EMBO J. 6:307-311) may be used; alternatively, plant promoterssuch as the small subunit of RUBISCO (Coruzzi et al., 1984, EMBO J.3:1671-1680; Broglie et al., 1984, Science 224:838-843) or heat shockpromoters, e.g., soybean hsp17.5-E or hsp17.3-B (Gurley et al., 1986,Mol. Cell. Biol. 6:559-565) may be used. These constructs can beintroduced into planleukocytes using Ti plasmids, Ri plasmids, plantvirus vectors, direct DNA transformation, microinjection,electroporation, etc. For reviews of such techniques see, e.g.,Weissbach & Weissbach, 1988, Methods for Plant Molecular Biology,Academic Press, NY, Section VIII, pp. 421-463; and Grierson & Corey,1988, Plant Molecular Biology, 2d Ed., Blackie, London, Ch. 7-9.

In one insect expression system that may be used to produce the peptidesof the invention, Autographa californica nuclear polyhidrosis virus(AcNPV) is used as a vector to express the foreign genes. The virusgrows in Spodoptera frugiperda cells. A coding sequence may be clonedinto non-essential regions (for example the polyhedron gene) of thevirus and placed under control of an AcNPV promoter (for example, thepolyhedron promoter). Successful insertion of a coding sequence willresult in inactivation of the polyhedron gene and production ofnon-occluded recombinant virus (i.e., virus lacking the proteinaceouscoat coded for by the polyhedron gene). These recombinant viruses arethen used to infect Spodoptera frugiperda cells in which the insertedgene is expressed. (e.g., see Smith et al., 1983, J. Virol. 46:584;Smith, U.S. Pat. No. 4,215,051). Further examples of this expressionsystem may be found in Current Protocols in Molecular Biology, Vol. 2,Ausubel et al., eds., Greene Publish. Assoc. & Wiley Interscience.

In mammalian host cells, a number of viral based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, a coding sequence may be ligated to an adenovirustranscription/translation control complex, e.g., the late promoter andtripartite leader sequence. This chimeric gene may then be inserted inthe adenovirus genome by in vitro or in vivo recombination. Insertion ina non-essential region of the viral genome (e.g., region E1 or E3) willresult in a recombinant virus that is viable and capable of expressingpeptide in infected hosts. (e.g., See Logan & Shenk, 1984, Proc. Natl.Acad. Sci. USA 81:3655-3659). Alternatively, the vaccinia 7.5 K promotermay be used, (see, e.g., Mackett et al., 1982, Proc. Natl. Acad. Sci.USA 79:7415-7419; Mackett et al., 1984, J. Virol. 49:857-864; Panicaliet al., 1982, Proc. Natl. Acad. Sci. USA 79:4927-4931).

Other expression systems for producing linear peptides of the inventionwill be apparent to those having skill in the art.

C. Tags or Markers

Tags and markers are frequently used to aid in purification ofcomponents or detection of biological molecules. Examples of biologicaltags include, but are not limited to, glutathione-S-transferase, maltosebinding protein, Immunoglobulin domains, Intein, Hemagglutinin epitopes,myc epitopes, etc. Examples of chemical tags include, but are notlimited to, biotin, gold, paramagnetic particles or fluorophores. Theseexamples can be used to identify the presence of proteins or compoundsthey are attached to or can be used by those skilled in the art topurify proteins or compounds from complex mixtures.

D. Purification of the Peptides and Peptide Analogues

The peptides and peptide analogues of the invention can be purified byart-known techniques such as high performance liquid chromatography, ionexchange chromatography, gel electrophoresis, affinity chromatographyand the like. The actual conditions used to purify a particular peptideor analogue will depend, in part, on factors such as net charge,hydrophobicity, hydrophilicity, etc., and will be apparent to thosehaving skill in the art. The purified peptides can be identified byassays based on their physical or functional properties, includingradioactive labeling followed by gel electrophoresis,radioimmuno-assays, ELISA, bioassays, and the like.

XII. Kits

The present invention also includes kits for carrying out the methods ofthe invention. A subject kit usually contains a first and a secondoncogenic HPV E6 binding partner. In most embodiments, the first bindingpartner is a PDZ domain polypeptide, and, the second binding partner isat least one antibody for E6. In some embodiments, the second bindingpartner is labeled with a detectable label. In other embodiments, asecondary labeling component, such as a detectably labeled secondaryantibody, is included. In some embodiments, a subject kit furthercomprises a means, such as a device or a system, for isolating oncogenicHPV E6 from the sample. The kit may optionally contain proteasomeinhibitor.

A subject kit can further include, if desired, one or more of variousconventional components, such as, for example, containers with one ormore buffers, detection reagents or antibodies. Printed instructions,either as inserts or as labels, indicating quantities of the componentsto be used and guidelines for their use, can also be included in thekit. In the present disclosure it should be understood that thespecified materials and conditions are important in practicing theinvention but that unspecified materials and conditions are not excludedso long as they do not prevent the benefits of the invention from beingrealized. Exemplary embodiments of the diagnostic methods of theinvention are described above in detail.

In a subject kit, the oncogenic E6 detection reaction may be performedusing an aqueous or solid substrate, where the kit may comprise reagentsfor use with several separation and detection platforms such as teststrips, sandwich assays, etc. In many embodiments of the test strip kit,the test strip has bound thereto a PDZ domain polypeptide thatspecifically binds the PL domain of an oncogenic E6 protein and capturesoncogenic E6 protein on the solid support. In some embodiments, the kitfurther comprises a detection antibody or antibodies, which is eitherdirectly or indirectly detectable, and which binds to the oncogenic E6protein to allow its detection. Kits may also include components forconducting western blots (e.g., pre-made gels, membranes, transfersystems, etc.); components for carrying out ELISAs (e.g., 96-wellplates); components for carrying out immunoprecipitation (e.g. proteinA); columns, especially spin columns, for affinity or size separation ofoncogenic E6 protein from a sample (e.g. gel filtration columns, PDZdomain polypeptide columns, size exclusion columns, membrane cut-offspin columns etc.).

Subject kits may also contain control samples containing oncogenic ornon-oncogenic E6, and/or a dilution series of oncogenic E6, where thedilution series represents a range of appropriate standards with which auser of the kit can compare their results and estimate the level ofoncogenic E6 in their sample. Such a dilution series may provide anestimation of the progression of any cancer in a patient. Fluorescence,color, or autoradiological film development results may also be comparedto a standard curves of fluorescence, color or film density provided bythe kit.

In addition to above-mentioned components, the subject kits typicallyfurther include instructions for using the components of the kit topractice the subject methods. The instructions for practicing thesubject methods are generally recorded on a suitable recording medium.For example, the instructions may be printed on a substrate, such aspaper or plastic, etc. As such, the instructions may be present in thekits as a package insert, in the labeling of the container of the kit orcomponents thereof (i.e., associated with the packaging or subpackaging)etc. In other embodiments, the instructions are present as an electronicstorage data file present on a suitable computer readable storagemedium, e.g. CD-ROM, diskette, etc. In yet other embodiments, the actualinstructions are not present in the kit, but means for obtaining theinstructions from a remote source, e.g. via the internet, are provided.An example of this embodiment is a kit that includes a web address wherethe instructions can be viewed and/or from which the instructions can bedownloaded. As with the instructions, this means for obtaining theinstructions is recorded on a suitable substrate.

Also provided by the subject invention is are kits including at least acomputer readable medium including programming as discussed above andinstructions. The instructions may include installation or setupdirections. The instructions may include directions for use of theinvention with options or combinations of options as described above. Incertain embodiments, the instructions include both types of information.

Providing the software and instructions as a kit may serve a number ofpurposes. The combination may be packaged and purchased as a means forproducing rabbit antibodies that are less immunogenic in a non-rabbithost than a parent antibody, or nucleotide sequences them.

The instructions are generally recorded on a suitable recording medium.For example, the instructions may be printed on a substrate, such aspaper or plastic, etc. As such, the instructions may be present in thekits as a package insert, in the labeling of the container of the kit orcomponents thereof (i.e., associated with the packaging orsubpackaging), etc. In other embodiments, the instructions are presentas an electronic storage data file present on a suitable computerreadable storage medium, e.g., CD-ROM, diskette, etc, including the samemedium on which the program is presented.

Methods of Determining if a Subject is Infected with an Oncogenic Strainof HPV

The present invention provides methods of detecting oncogenic HPV E6protein in a sample and finds utility in diagnosing HPV infection in asubject. In many embodiment, a biological sample is obtained from asubject, and, the presence of oncogenic HPV E6 protein in the sample isdetermined. The presence of a detectable amount of oncogenic HPV E6protein in a sample indicates indicates that the individual is infectedwith a oncogenic strain of HPV. In other embodiments, the level ofoncogenic HPV E6 protein in a biological sample is determined, andcompared to the amount of a control in the sample. The relative amountof oncogenic HPV E6 protein in a sample indicates the severity of theinfection by HPV.

The methods generally involve two binding partners of oncogenic HPV E6protein, one of which is a PDZ domain polypeptide, as described above.In general, the methods involve a) isolating the oncogenic HPV E6protein from a sample using one of the binding partners, and b)detecting the oncogenic HPV E6 protein with the other binding partner.

Isolating Oncogenic HPV E6 Protein

In general, methods of the invention involve at least partiallyseparating (i.e., isolating) native oncogenic HPV E6 protein from otherproteins in a sample. This separation is usually achieved using a firstbinding partner for the oncogenic HPV E6. In many embodiments, the firstbinding partner is a PDZ domain polypeptide, or, in other embodiments ananti-HPV E6 antibody or mixture of antibodies.

In certain embodiments, one of the oncogenic HPV E6 binding partners isbound, directly or via a linker, to an insoluble support. Insolublesupports are known in the art and include, but are not limited to, abead (e.g., magnetic beads, polystyrene beads, and the like); amembrane; and the like. In one non-limiting example, a PDZ domainpolypeptide is bound to a magnetic bead. The PDZ domain polypeptidebound to the magnetic bead is contacted with the sample, and, after acomplex is formed between the antibody and any E6 protein in the sample,a magnetic field is applied, such that the complex is removed from thesample. Where the PDZ domain polypeptide is bound to an insolublesupport, such as a membrane, E6 protein bound to the PDZ domainpolypeptide is removed from the sample by removing the membrane, or bytransferring the sample to a separate container. Where the PDZ domainpolypeptide is bound to a bead, the E6 protein bound to the bead isremoved from the sample by centrifugation or filtration. Suchembodiments are envisioned using a different E6 binding partner, e.g.,an anti-E6 antibody.

In general, a suitable separation means is used with a suitable platformfor performing the separation. For example, where oncogenic HPV E6 isseparated by binding to PDZ domain polypeptides, the separation isperformed using any of a variety of platforms, including, but notlimited to, affinity column chromatography, capillary action or lateralflow test strips, immunoprecipitation, etc.

In many embodiments, oncogenic HPV E6 is separated from other proteinsin the sample by applying the sample to one end of a test strip, andallowing the proteins to migrate by capillary action or lateral flow.Methods and devices for lateral flow separation, detection, andquantitation are known in the art. See, e.g., U.S. Pat. Nos. 5,569,608;6,297,020; and 6,403,383. In these embodiments, a test strip comprises,in order from proximal end to distal end, a region for loading thesample (the sample-loading region) and a test region containing anoncogenic E6 protein binding partner, e.g., a region containing an PDZdomain polypeptide or, in other embodiments, a region containing ananti-E6 antibody. The sample is loaded on to the sample-loading region,and the proximal end of the test strip is placed in a buffer oncogenicE6 protein is captured by the bound antibody in the first test region.Detection of the captured oncogenic E6 protein is carried out asdescribed below. For example, detection of captured E6 proteins iscarried out using detectably labeled antibody specific for an epitope ofE6 proteins that is common to all oncogenic E6 proteins, or a mixture ofantibodies that can, together, bind to all oncogenic E6 proteins. Inalternative embodiments, an E6 antibody may be present in the testregion and detection of oncogenic E6 bound to the E6 antibody uses alabeled PDZ domain polypeptide.

Detecting and Quantitating Oncogenic E6 Protein

Once oncogenic E6 protein is separated from other proteins in thesample, oncogenic E6 protein is detected and/or the level or amount ofoncogenic E6 protein is determined (e.g., measured). As discussed above,oncogenic E6 protein is generally detected using a binding partner, e.g.an antibody or antibodies specific to E6, or a PDZ domain polypeptide.

Detection with a specific antibody is carried out using well-knownmethods. In general, the binding partner is detectably labeled, eitherdirectly or indirectly. Direct labels include radioisotopes (e.g., ¹²⁵I;³⁵S, and the like); enzymes whose products are detectable (e.g.,luciferase, β-galactosidase, horse radish peroxidase, and the like);fluorescent labels (e.g., fluorescein isothiocyanate, rhodamine,phycoerytlrin, and the like); fluorescence emitting metals, e.g., ¹⁵²Eu,or others of the lanthanide series, attached to the antibody throughmetal chelating groups such as EDTA; chemiluminescent compounds, e.g.,luminol, isoluminol, acridinium salts, and the like; bioluminescentcompounds, e.g., luciferin; fluorescent proteins; and the like.Fluorescent proteins include, but are not limited to, a greenfluorescent protein (GFP), including, but not limited to, a “humanized”version of a GFP, e.g., wherein codons of the naturally-occurringnucleotide sequence are changed to more closely match human codon bias;a GFP derived from Aequoria victoria or a derivative thereof, e.g., a“humanized” derivative such as Enhanced GFP, which are availablecommercially, e.g., from Clontech, Inc.; a GFP from another species suchas Renilla reniformis, Renilla mullei, or Ptilosarcus guernyi, asdescribed in, e.g., WO 99/49019 and Peelle et al. (2001) J. ProteinChem. 20:507-519; “humanized” recombinant GFP (hrGFP) (Stratagene); anyof a variety of fluorescent and colored proteins from Anthozoan species,as described in, e.g., Matz et al. (1999) Nature Biotechnol. 17:969-973;and the like.

Indirect labels include second antibodies specific for E6-specificantibodies, wherein the second antibody is labeled as described above;and members of specific binding pairs, e.g., biotin-avidin, and thelike.

In some embodiments, a level of oncogenic E6 is quantitated.Quantitation can be carried out using any known method, including, butnot limited to, enzyme-linked immunosorbent assay (ELISA);radioimmunoassay (RIA); and the like. In general, quantitation isaccomplished by comparing the level of expression product detected inthe sample with a standard curve.

In some embodiments, oncogenic HPV E6 is separated on a test strip, asdescribed above. In these embodiments, oncogenic HPV E6 is detectedusing a detectably labeled binding partner that binds oncogenic HPV E6.Oncogenic HPV E6 may be quantitated using a reflectancespectrophotometer, or by eye, for example.

Biological Samples

Biological samples to be analyzed using the methods of the invention areobtained from any mammal, e.g., a human or a non-human animal model ofHPV. In many embodiments, the biological sample is obtained from aliving subject.

In some embodiments, the subject from whom the sample is obtained isapparently healthy, where the analysis is performed as a part of routinescreening. In other embodiments, the subject is one who is susceptibleto HPV, (e.g., as determined by family history; exposure to certainenvironmental factors; etc.). In other embodiments, the subject hassymptoms of HPV (e.g., cervical warts, or the like). In otherembodiments, the subject has been provisionally diagnosed as having HPV(e.g. as determined by other tests based on e.g., PCR).

The biological sample may be derived from any tissue, organ or group ofcells of the subject. In some embodiments a cervical scrape, biopsy, orlavage is obtained from a subject.

In some embodiments, the biological sample is processed, e.g., to removecertain components that may interfere with an assay method of theinvention, using methods that are standard in the art. In someembodiments, the biological sample is processed to enrich for proteins,e.g., by salt precipitation, and the like. In certain embodiments, thesample is processed in the presence proteasome inhibitor to inhibitdegradation of the E6 protein.

In the assay methods of the invention, in some embodiments, the level ofE6 protein in a sample may be quantified and/or compared to controls.Suitable control samples are from individuals known to be healthy, e.g.,individuals known not to have HPV. Control samples can be fromindividuals genetically related to the subject being tested, but canalso be from genetically unrelated individuals. A suitable controlsample also includes a sample from an individual taken at a time pointearlier than the time point at which the test sample is taken, e.g., abiological sample taken from the individual prior to exhibiting possiblesymptoms of HPV.

Utility

The methods of the instant invention are useful for a variety ofdiagnostic analyses. The instant methods are useful for diagnosinginfection by an oncogenic strain of HPV in an individual; fordetermining the likelihood of having cancer; for determining a patient'sresponse to treatment for HPV; for determining the severity of HPVinfection in an individual; and for monitoring the progression of HPV inan individual.

The subject methods may generally be performed on biological samplesfrom living subjects. A particularly advantageous feature of theinvention is that the methods can simultaneously detect, in onereaction, all known oncogenic strains of HPV.

EXAMPLE 1 Sequence Analysis of HPV E6 Proteins to Determine OncogenicPotential

PDZ proteins are known to bind certain carboxyl-terminal sequences ofproteins (PLs). PL sequences that bind PDZ domains are predictable, andhave been described in greater detail in U.S. patent application Ser.Nos. 09/710,059, 09/724,553 and 09/688,017. One of the major classes ofPL motifs is the set of proteins terminating in the sequences-X-(S/T)-X-(V/I/L). We have examined the C-terminal sequences of E6proteins from a number of HPV strains. All of the strains determined tobe oncogenic by the National Cancer Institute exhibit a consensus PDZbinding sequence. Those E6 proteins from papillomavirus strains that arenot cancerous lack a sequence that would be predicted to bind to PDZdomains, thus suggesting that interaction with PDZ proteins is aprerequisite for causing cancer in humans. This correlation betweenpresence of a PL and ability to cause cancer is 100% in the sequencesexamined (Table 3A). In theory, with the disclosed PL consensussequences from the patents listed supra, new variants of HPVs can beassessed for their ability to bind PDZ proteins and oncogenicity can bepredicted on the basis of whether a PL is present. Earlier this year,five new oncogenic strains of Human papillomavirus were identified andtheir E6 proteins sequenced. As predicted, these proteins all contain aPL consensus sequence (Table 3B).

TABLE 3A Correlation of E6 PDZ-ligands and oncogenicity E6 C-terminal PLHPV strain sequence yes/no oncogenic Seq ID No HPV 4 GYCRNCIRKQ No No221 HPV 11 WTTCMEDLLP No No 222 HPV 20 GICRLCKHFQ No No 223 HPV 24KGLCRQCKQI No No 224 HPV 28 WLRCTVRIPQ No No 225 HPV 36 RQCKHFYNDW No No226 HPV 48 CRNCISHEGR No No 227 HPV 50 CCRNCYEHEG No No 228 HPV 16SSRTRRETQL Yes Yes 229 HPV 18 RLQRRRETQV Yes Yes 230 HPV 31 WRRPRTETQVYes Yes 231 HPV 35 WKPTRRETEV Yes Yes 232 HPV 30 RRTLRRETQV Yes Yes 233HPV 39 RRLTRRETQV Yes Yes 234 HPV 45 RLRRRRETQV Yes Yes 235 HPV 51RLQRRNETQV Yes Yes 236 HPV 52 RLQRRRVTQV Yes Yes 237 HPV 56 TSREPRESTVYes Yes 238 HPV 59 QRQARSETLV yes Yes 239 HPV 58 RLQRRRQTQV Yes Yes 240HPV 33 RLQRRRETAL Yes Yes 241 HPV 66 TSRQATESTV Yes  Yes* 242 HPV 68RRRTRQETQV Yes Yes 243 HPV 69 RRREATETQV Yes Yes 244 TABLE 3A: E6C-terminal sequences and oncogenicity. HPV variants are listed at theleft. Sequences were identified from Genbank sequence records. PL Yes/Nowas defined by a match or non-match to the consenses determined at ArborVita and by Songyang et al. -X- (S/T)-X-(V/I/L). Oncogenicity datacollected from National Cancer Institute. *Only found in oncogenicstrains co-transfected with other oncogenic proteins.

TABLE 3B Correlation of recently identified oncogenic E6 proteins E6C-terminal PL HPV strain sequence yes/no oncogenic Seq ID No HPV 26RPRRQTETQV Yes Yes 245 HPV 53 RHTTATESAV Yes Yes 246 HPV 66 TSRQATESTVYes Yes 247 HPV 73 RCWRPSATVV Yes Yes 248 HPV 82 PPRQRSETQV Yes Yes 249TABLE 3B: E6 C-terminal sequences and oncogenicity. HPV variants arelisted at the left. Sequences were identified from Genbank sequencerecords. PL Yes/No was defined by a match or non-match to the consensesdetermined at Arbor Vita and by Songyang et al. -X- (S/T)-X-(V/I/L).Oncogenicity data on new strains collected from N Engl J Med 2003;348:518-527.

These tables provide a classification of the HPV strains based on thesequence of the C-terminal four amino acids of the E6 protein encoded bythe HPV genome. The 21 oncogenic strains of HPV fall into one of 10classes, and HPV strains not specifically listed above may also fallinto these classes. As such, it is desirable to detect HPV strains fromall 10 classes: the instant methods provide such detection.

EXAMPLE 2 Identification of PDZ Domains that Interact with the C-Terminiof Oncogenic E6 Proteins

In order to determine the PDZ domains that can be used to detectoncogenic E6 proteins in a diagnostic assay, the ‘G assay’ (describedsupra) was used to identify interactions between E6 PLs and PDZ domains.Peptides were synthesized corresponding to the C-terminal amino acidsequences of E6 proteins from oncogenic strains of human papillomavirus.These peptides were assessed for the ability to bind PDZ domains usingthe G-assay described above and PDZ proteins synthesized from theexpression constructs described in greater detail in U.S. patentapplication Ser. Nos. 09/710,059, 09/724,553 and 09/688,017. Results ofthese assays that show a high binding affinity are listed in Table 4below.

As we can see below, there a large number of PDZ domains that bind someof the oncogenic E6 proteins. However, only the second PDZ domain fromMAGI-1 seems to bind all of the oncogenic E6 PLs tested. The PDZ domainof TIP-1 binds all but one of the oncogenic E6 PLs tested, and may beuseful in conjunction with MAGI-1 domain 2 for detecting the presence ofoncogenic E6 proteins.

In a similar manner, peptides corresponding to the C-terminal ends ofseveral non-oncogenic E6 proteins were tested with the G-assay. None ofthe peptides showed any affinity for binding PDZ domains.

TABLE 4 higher affinity interactions between HPV E6 PLs and PDZ domainsHPV PDZ binding partner HPV PDZ binding partner strain (signal 4 and 5of 0-5) strain (signal 4 and 5 of 0-5) HPV 35 Atrophin-1 interact. prot.HPV 33 Magi1 (PDZ #2) (TEV) (PDZ # 1, 3, 5) (TAL) TIP1 Magi1 (PDZ # 2,3, 4, 5) DLG1 Lim-Ril Vartul (PDZ #1) FLJ 11215 KIAA 0807 MUPP-1 (PDZ#10) KIAA 1095 (Semcap3) KIAA 1095 (PDZ #1) (PDZ #1) PTN-4 KIAA 1934(PDZ #1) INADL (PDZ #8) NeDLG (PDZ #1, 2) Vartul (PDZ # 1, 2, 3) Ratouter membrane Syntrophin-1 alpha (PDZ #1) Syntrophin gamma-1 PSD 95 TAXIP2 (PDZ #3 and 1-3) KIAA 0807 KIAA 1634 (PDZ #1) DLG1 (PDZ1, 2) NeDLG(1, 2, 3,) Sim. Rat outer membrane (PDZ #1) MUPP-1 (PDZ #13) PSD 95 (1,2, 3) HPV 58 Atrophin-1 interact. prot. HPV 66 DLG1 (PDZ #1, 2) (TQV)(PDZ #1) (STV) NeDLG (PDZ #2) Magi1 (PDZ #2) PSD 95 (PDZ #1, 2, 3) DLG1(PDZ1, 2) Magi1 (PDZ #2) DLG2 (PDZ #2) KIAA 0807 KIAA 0807 KIAA 1634(PDZ #1) KIAA 1634 (PDZ #1) DLG2 (PDZ #2) NeDLG (1, 2) Rat outermembrane Sim. Rat outer membrane (PDZ #1) (PDZ #1) NeDLG (1, 2) PSD 95(1, 2, 3) TIP-1 INADL (PDZ #8) TIP-1 HPV 16* TIP-1 HPV 52 Magi1 (PDZ #2)(TQL) Magi1 (PDZ #2) (TQV) HPV 18* TIP1 (TQV) Magi 1 (PDZ #2) Table 4:Interactions between the E6 C-termini of several HPV variants and humanPDZ domains. HPV strain denotes the strain from which the E6 C-terminalpeptide sequence information was taken. Peptides used in the assayvaried from 18 to 20 amino acids in length, and the terminal fourresidues arelisted in parenthesis. Names to the right of each HPV E6variant denote the human PDZ domain(s) (with domain number inparenthesis for proteins with multiple PDZ domains) that saturatedbinding with the E6 peptide in the G assay (See Description of theInvention). *denotes that the PDZ domains of hDlg1 were not testedagainst these proteins yet due to limited material, although both havebeen shown to bind hDlg1 in the literature.

EXAMPLE 3 Generation of Eukaryotic Expression Constructs Bearing DNAFragments that Encode HPV E6 Genes or Portions of HPV E6 Genes

This example describes the cloning of HPV E6 genes or portions of HPV E6genes into eukaryotic expression vectors in fusion with a number ofprotein tags, including but not limited to Glutathione S-Transferase(GST), Enhanced Green Fluorescent Protein (EGFP), or Hemagglutinin (HA).

A. Strategy cDNA fragments were generated by RT-PCR from HPV cell line(cervical epidermoid carcinoma, ATCC# CRL-1550 and CRL-1595 for HPV E616 and 18, respectively) derived RNA, using random (oligo-nucleotide)primers (Invitrogen Cat.# 48190011). DNA fragments corresponding to HPVE6 were generated by standard PCR, using above purified cDNA fragmentsand specific primers (see Table 5). Primers used were designed to createrestriction nuclease recognition sites at the PCR fragment's ends, toallow cloning of those fragments into appropriate expression vectors.Subsequent to PCR, DNA samples were submitted to agarose gelelectrophoresis. Bands corresponding to the expected size were excised.DNA was extracted by Sephaglas Band Prep Kit (Amersham Pharmacia Cat#27-9285-01) and digested with appropriate restriction endonuclease.Digested DNA samples were purified once more by gel electrophoresis,according to the same protocol used above. Purified DNA fragments werecoprecipitated and ligated with the appropriate linearized vector. Aftertransformation into E. coli, bacterial colonies were screened by colonyPCR and restriction digest for the presence and correct orientation ofinsert. Positive clones were innoculated in liquid culture for largescale DNA purification. The insert and flanking vector sites from thepurified plasmid DNA were sequenced to ensure correct sequence offragments and junctions between the vectors and fusion proteins.

B. Vectors:

Cloning vectors were pGEX-3× (Amersham Pharmacia #27-4803-01), MIE (aderivative of MSCV, containing IRES and EGFP, generated by recombinantDNA technology), pmKit, pcDNA3.1 (Invitrogen, modified to include a HAtag upstream of the cloning site) and pMAL (New England Biolabs Cat#N8076S, polylinker modified in house to include Bam-H1 and EcoR1 sites).

DNA fragments containing the ATG-start codon and the TAG-stop codon ofHPV E6 were cloned into pGEX3×. HPV E6 genes, and 3′ truncated (APL)versions, were subsequently cloned into MIE (MSCV-IRES-EGFP) vector,pcDNA-HA vector, and pmKit vector, using the purified HPV E6-pGEX3xfusion plasmid as the PCR template, and using the same purificationprotocols as listed above. Truncated versions of HPV E6 have a stopcodon inserted after the −3 position amino acid, so as to delete thelast three amino acids from the coding region of the gene.

C. Constructs:

Primers used to generate DNA fragments by PCR are listed in Table 5. PCRprimer combinations and restriction sites for insert and vector arelisted below.

TABLE 5 Primers used in cloning of HPV E6 into representative expressionvectors. ID# Seq (Primer Name) Primer Sequence Description ID 2548AAAAGATCTACAAT Forward (5′ to 3′) primer corresponding to 250 (1054EF)ACTATGGCGC HPV E6 18, generates a Bgl II site. Used for cloning intopGEX3x. 2549 AGGGAATTCCAGAC Reverse (3′ to 5′) primer corresponding to251 (1058ER) TTAATATTATAC HPV E6 18, generates an EcoR1 site. Used forcloning into pGEX3x. 2542 AAAGGATCCATTTT Forward (5′ to 3′) primercorresponding to 252 (1050EF) ATGCACCAAAAG HPV E6 16, generates a BamH1site. Used for cloning into pGEX3x. 2543 ATGGAATTCTATCTC Reverse (3′ to5′) primer corresponding to 253 (1051ER) CATGCATGATTAC HPV E6 16,generates an EcoR1 site. Used for cloning into pGEX3x. 2563GAGGAATTCACCAC Forward (5′ to 3′) primer corresponding to 254 (1071EF)AATACTATGGCG HPV E6 18, generates an EcoR1 site. Used for cloning intoMIE. 2564 AGGAGATCTCATAC Reverse (3′ to 5′) primer corresponding to 255(1072ER) TTAATATTATAC HPV E6 18, generates a Bgl II site. Used forcloning into MIE. 2565 TTGAGATCTTCAGC Reverse (3′ to 5′) primercorresponding to 256 (1073ERPL) GTCGTTGGAGTCG HPV E6 18 ΔPL, generates aBgl II site. Used for cloning into MIE. 2560 AAAGAATTCATTTT Forward(5′ to 3′) primer corresponding to 257 (1074EF) ATGCACCAAAAG HPV E6 16,generates an EcoR1 site. Used for cloning into MIE. 2561 ATGGGATCCTATCTCReverse (3′ to 5′) primer corresponding to 258 (1075ER) CATGCATGATTACHPV E6 16, generates a BamH1 site. Used for cloning into MIE. 2562CTGGGATCCTCATC Reverse (3′ to 5′) primer corresponding to 259 (1076ERPL)AACGTGTTTCTTGATG HPV E6 16 ΔPL, generates a BamH1 site. ATC Used forcloning into MIE. 2603 AAGAAAGCTTTTTA Forward (5′ to 3′) primercorresponding to 260 (1080EF) TGCACCAAAAGAG HPV E6 16, generates A HindIII site. Used for cloning into pcDNA-HA. 2604 AATCAAGCTTTTATCT Reverse(3′ to 5′) primer corresponding to 261 (1081ER) CCATGCATGATTAC HPV E616, generates a Hind III site. Used for cloning into pcDNA-HA. 2605GCTGAAGCTTTCAA Reverse (3′ to 5′) primer corresponding to 262 (1082ERPL)CGTGTTCTTGATGAT HPV E6 16 ΔPL, generates a Hind III site. C Used forcloning into pcDNA-HA. 2606 AAGCGTCGACTTTA Forward (5′ to 3′) primercorresponding to 263 (1083EF) TGCACCAAAAGAG HPV E6 16, generates a Sal Isite. Used for cloning into pmKit. 2607 AATGCTCGAGTATC Reverse (3′ to5′) primer corresponding to 264 (1084ER) TCCATGCATGATTAC HPV E6 16,generates a Xho I site. Used for cloning into pmKit. 2608 GCTGCTCGAGTCAAReverse (3′ to 5′) primer corresponding to 265 (1085ERPL)CGTGTTCTTGATGAT HPV E6 16 ΔPL, generates a Xho I site. Used C forcloning into pmKit. 2612 AGAAGTCGACCACA Forward (5′ to 3′) primercorresponding to 266 (1086EF) ATACTATGGCGC HPV E6 18, generates a Sal Isite. Used for cloning into pmKit. 2613 TAGGCTCGAGCATA Reverse (3′ to5′) primer corresponding to 267 (1087ER) CTTAATATTATAC HPV E6 18,generates a Xho I site. Used for cloning into pmKit. 2614 CTTGCTCGAGTCAGReverse (3′ to 5′) primer corresponding to 268 (1088ERPL) CGTCGTTGGAGTCGHPV E6 18 ΔPL , generates a Xho I site. Used for cloning into pmKit.2615 AGAAAAGCTTCACA Forward (5′ to 3′) primer corresponding to 269(1089EF) ATACTATGGCGC HPV E6 18, generates A Hind III site. Used forcloning into pcDNA-HA. 2616 TAGAAGCTTGCATA Reverse (3′ to 5′) primercorresponding to 270 (1090ER) CTTAATATTATAC HPV E6 18, generates a HindIII site. Used for cloning into pcDNA-HA. 2617 CTTGAAGCTTTCAGC Reverse(3′ to 5′) primer corresponding to 271 (1091ERPL) GTCGTTGAGGTCG HPV E618 ΔPL, generates a Hind III site Used for cloning into pcDNA-HA.

1. Human Papillomavirus (HPV) E6 16

Acc#:-------------

GI#:4927719

Construct: HPV E6 16WT-pGEX-3×

Primers: 2542 & 2543

Vector Cloning Sites(5′/3′): Bam H1/EcoR1

Insert Cloning Sites(5′/3′): BamH1/EcoR1

pGEX-3× contains GST to the 5′ end (upstream) of the cloning site

Construct: HPV E6 16WT-MIE

Primers: 2560 & 2561

Vector Cloning Sites(5′/3′): EcoR1/BamH1

Insert Cloning Sites(5′/3′): EcoR1/BamH1

MIE contains IRES and EGFP to the 3′ end (downstream) of the cloningsite

Construct: HPV E6 16ΔPL-MIE

Primers: 2560 & 2562

Vector Cloning Sites(5′/3′): EcoR1/BamH1

Insert Cloning Sites(5′/3′): EcoR1/BamH1

MIE contains IRES and EGFP to the 3′ end (downstream) of the cloningsite

Construct: HPV E6 16WT-pcDNA3.1-HA

Primers: 2603 & 2604

Vector Cloning Sites(5′/3′): Hind III/Hind III

Insert Cloning Sites(5′/3′): Hind III/Hind III

pcDNA3.1 (modified) contains HA to the 5′ end (upstream) of the cloningsite

Construct: HPV E6 16ΔPL-pcDNA3.1-HA

Primers: 2603 & 2605

Vector Cloning Sites(5′/3′): Hind III/Hind III

Insert Cloning Sites(5′/3′): Hind III/Hind III

pcDNA3.1 (modified) contains HA to the 5′ end (upstream) of the cloningsite

Construct: HPV E6 16WT-pmKit

Primers: 2606 & 2607

Vector Cloning Sites(5′/3′): Sal I/Xho I

Insert Cloning Sites(5′/3′): Sal Ixho I

Construct: HPV E6 16ΔPL-pmkit

Primers: 2606 & 2608

Vector Cloning Sites(5′/3′): Sal I/Xho I

Insert Cloning Sites(5′/3′): Sal I/Xho I

2. Human Papillomavirus (HPV) E6 18

Acc#:-------------

GI#:-------------

Construct: HPV E6 18WT-pGEX-3×

Primers: 2548 & 2549

Vector Cloning Sites(5′/3′): Bam H1/EcoR1

Insert Cloning Sites(5′/3′): Bgl II/EcoR1

pGEX-3× contains GST to the 5′ end (upstream) of the cloning site

Construct: HPV E6 18WT-MIE

Primers: 2563 & 2564

Vector Cloning Sites(5′/3′): EcoR1/BamH1

Insert Cloning Sites(5′/3′): EcoR1/Bgl II

MIE contains IRES and EGFP to the 3′ end (downstream) of the cloningsite

Construct: HPV E6 18ΔPL-MIE

Primers: 2563 & 2565

Vector Cloning Sites(5′/3′): EcoR1/BamH1

Insert Cloning Sites(5′/3′): EcoR1/Bgl II

MIE contains IRES and EGFP to the 3′ end (downstream) of the cloningsite

Construct: HPV E6 18WT-pcDNA3.1-HA

Primers: 2615 & 2616

Vector Cloning Sites(5′/3′): Hind III/Hind III

Insert Cloning Sites(5′/3′): Hind III/Hind III

pcDNA3.1 (modified) contains HA to the 5′ end (upstream) of the cloningsite

Construct: HPV E6 18ΔPL-pcDNA3.1-HA

Primers: 2615 & 2617

Vector Cloning Sites(5′/3′): Hind III/Hind III

Insert Cloning Sites(5′/3′): Hind III/Hind III

pcDNA3.1 (modified) contains HA to the 5′ end (upstream) of the cloningsite

Construct: HPV E6 18WT-pmKit

Primers: 2612 & 2613

Vector Cloning Sites(5′/3′): Sal I/Xho I

Insert Cloning Sites(5′/3′): Sal I/Xho I

Construct: HPV E6 18ΔPL-pmKit

Primers: 2612 & 2614

Vector Cloning Sites(5′/3′): Sal I/Xho I

Insert Cloning Sites(5′/3′): Sal I/Xho I

D. GST Fusion Protein Production and Purification

The constructs using pGEX-3× expression vector were used to make fusionproteins according to the protocol outlined in the GST Fusion System,Second Edition, Revision 2, Pharmacia Biotech. Method II and wasoptimized for a 1 L LgPP.

Purified DNA was transformed into E. coli and allowed to grow to anOD₆₀₀ of 0.4-0.8 (600λ). Protein expression was induced for 1-2 hours byaddition of IPTG to cell culture. Cells were harvested and lysed. Lysatewas collected and GS4B beads (Pharmacia Cat# 17-0756-01) were added tobind GST fusion proteins. Beads were isolated and GST fusion proteinswere eluted with GEB II. Purified proteins were stored in GEB II at −80°C.

Purified proteins were used for ELISA-based assays and antibodyproduction.

EXAMPLE 4 Generation of Eukaryotic Expression Constructs Bearing DNAFragments that Encode PDZ Domain Containing Genes or Portions of PDZDomain Genes

This example describes the cloning of PDZ domain containing genes orportions of PDZ domain containing genes were into eukaryotic expressionvectors in fusion with a number of protein tags, including but notlimited to Glutathione S-Transferase (GST), Enhanced Green FluorescentProtein (EGFP), or Hemagglutinin (HA).

A. Strategy

DNA fragments corresponding to PDZ domain containing genes weregenerated by RT-PCR from RNA from a library of individual cell lines(CLONTECH Cat# K4000-1) derived RNA, using random (oligo-nucleotide)primers (Invitrogen Cat.# 48190011). DNA fragments corresponding to PDZdomain containing genes or portions of PDZ domain containing genes weregenerated by standard PCR, using above purified cDNA fragments andspecific primers (see Table 6). Primers used were designed to createrestriction nuclease recognition sites at the PCR fragment's ends, toallow cloning of those fragments into appropriate expression vectors.Subsequent to PCR, DNA samples were submitted to agarose gelelectrophoresis. Bands corresponding to the expected size were excised.DNA was extracted by Sephaglas Band Prep Kit (Amersham PharmaciaCat#27-9285-01) and digested with appropriate restriction endonuclease.Digested DNA samples were purified once more by gel electrophoresis,according to the same protocol used above. Purified DNA fragments werecoprecipitated and ligated with the appropriate linearized vector. Aftertransformation into E. coli, bacterial colonies were screened by colonyPCR and restriction digest for the presence and correct orientation ofinsert. Positive clones were innoculated in liquid culture for largescale DNA purification. The insert and flanking vector sites from thepurified plasmid DNA were sequenced to ensure correct sequence offragments and junctions between the vectors and fusion proteins.

B. Vectors:

All PDZ domain-containing genes were cloned into the vector pGEX-3×(Amersham Pharmacia #27-4803-01, Genemed Acc#U13852, GI#595717),containing a tac promoter, GST, Factor Xa, β-lactamase, and lacrepressor.

The amino acid sequence of the pGEX-3× coding region including GST,Factor Xa, and the multiple cloning site is listed below. Note thatlinker sequences between the cloned inserts and GST-Factor Xa varydepending on the restriction endonuclease used for cloning. Amino acidsin the translated region below that may change depending on theinsertion used are indicated in small caps, and are included as changedin the construct sequence listed in (C).

aa 1-aa 232:

(SEQ ID NO: 272) MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEJSMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKI(RIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDLIEGRgipgnss

In addition, TAX Interacting Protein 1 (TIP1), in whole or part, wascloned into many other expression vectors, including but not limited toCD5γ, PEAKIO (both provided by the laboratory of Dr, Brian Seed atHarvard University and generated by recombinant DNA technology,containing an IgG region), and MIN (a derivative of MSCV, containingIRES and NGFR, generated by recombinant DNA technology).

C. Constructs:

Primers used to generate DNA fragments by PCR are listed in Table 6. PCRprimer combinations and restriction sites for insert and vector arelisted below, along with amino acid translation for insert andrestriction sites. Non-native amino acid sequences are shown in lowercase.

TABLE 6 Primers used in cloning of DLG 1 (domain 2 of 3), MAGI 1 (domain2 of 6), and TIP1 into representative expression vectors. ID# Seq(Primer Name) Primer Sequence Description ID 1928 AATGGGGATCCAGC Forward(5′ to 3′) primer corresponding to 273 (654DL1 2F) TCATTAAAGG DLG 1,domain 2 of 3. Generates a Bam H1 site upstream (5′) of the PDZboundary. Used for cloning into pGEX-3X. 1929 ATACATACTTGTGG Reverse(3′ to 5′) primer corresponding to 274 (655DL1 2R) AATTCGCCAC DLG 1,domain 2 of 3. Generates an EcoR1 site downstream (3′) of the PDZboundary. Used for cloning into pGEX-3X. 1453 CACGGATCCCTTCTG Forward(5′ to 3′) primer corresponding to 275 (435BAF) AGTTGAAAGGC MAGI 1,domain 2 of 6. Generates a BamH1 site upstream (5′) of the PDZ boundary.Used for cloning into pGEX-3X. 1454 TATGAATTCCATCTG Reverse (3′ to 5′)primer corresponding to 276 (436BAR) GATCAAAAGGCAAT MAGI 1, domain 2 of6. Generates an EcoR1 G site downstream (3′) of the PDZ boundary. Usedfor cloning into pGEX-3X. 399 (86TAF) CAGGGATCCAAAGA Forward (5′ to 3′)primer corresponding to 277 GTTGAAATTCACAA TIP1. Generates a Bam H1 siteupstream (5′) GC of the PDZ boundary. Used for cloning into pGEX-3X. 400(87TAR) ACGGAATTCTGCAG Reverse (3′ to 5′) primer corresponding to 278CGACTGCCGCGTC TIP1. Generates an EcoR1 site downstream (3′) of the PDZboundary. Used for cloning into pGEX-3X. 1319 (TIP AGGATCCAGATGTCForward (5′ to 3′) primer corresponding to 279 G5-1) CTACATCCC TIP1.Generates a Bam HI site upstream (5′) of the start codon. Used forcloning into pGEX-3X. 1320 (TIP GGAATTCATGGACT Reverse (3′ to 5′) primercorresponding to 280 G3-1) GCTGCACGG TIP1. Generates an EcoR1 sitedownstream (3′) of the stop codon. Used for cloning into pGEX-3X. 2753AGAGAATTCTCGAG Forward (5′ to 3′) primer corresponding to 281 (1090TIF)ATGTCCTACATCCC TIP1. Generates an EcoR1 site upstream (5′) of the startcodon. Used for cloning into MIN. 2762 TGGGAATTCCTAGG Reverse (3′ to 5′)primer corresponding to 282 (1117TIR) ACAGCATGGACTG TIP1. Generates anEcoR1 site downstream (3′) of the stop codon. Used for cloning into MIN.2584 CTAGGATCCGGGCC Forward (5′ to 3′) primer corresponding to 283(1080TIF) AGCCGGTCACC TIP1. Generates a Bam H1 site upstream (5′) of thePDZ boundary. Used for cloning into PEAK10 or CD5γ. 2585 GACGGATCCCCCTGReverse (3′ to 5′) primer corresponding to 284 (1081TIR) CTGCACGGCCTTCTGTIP1. Generates a Bam H1 site downstream (3′) of the PDZ boundary. Usedfor cloning into PEAK10 or CD5γ. 2586 GACGAATTCCCCTG Reverse (3′ to 5′)primer corresponding to 285 (1082T1R) CTGCACGGCCTTTCTG TIP1. Generatesan EcoR1 site downstream (3′) of the PDZ boundary. Used for cloning intoPEAK10 or CD5γ. 2587 CTAGAATTCGGGCC Forward (5′ to 3′) primercorresponding to 286 (1083T1F) AGCCGGTCACC TIP1. Generates an Eco R1site upstream (5′) of the PDZ boundary. Used for cloning into PEAK10 orCD5γ.1. DLG 1, PDZ domain 2 of 3:

Acc#:U13897

GI#:558437

-   -   Construct: DLG 1, PDZ domain 2 of 3-pGEX-3×        -   Primers: 1928 & 1929        -   Vector Cloning Sites(5′/3′): Bam H1/EcoR1        -   Insert Cloning Sites(5′/3′): BamH1/EcoR1        -   aa 1-aa88

(SEQ ID NO: 287) giqLIKGPKGLGFSIAGGVGNQHIPGDNSIYVTKIIEGGAAHKDGKLQIGDKLLAVNNVCLEEVTHEEAVTALKNTSDFVYLKVAnss2. MAGI 1, PDZ domain 2 of 6:

Acc#:AB010894

GI#:3370997

-   -   Construct: MAGI 1, PDZ domain 2 of 6-pGEX-3×        -   Primers: 1453 & 1454        -   Vector Cloning Sites(5′/3′): Bam H1/EcoR1        -   Insert Cloning Sites(5′/3′): BamH1/EcoR1        -   aa 1-aa 108

(SEQ ID NO: 288) giPSELKGKFIHTKLRKSSRGFGFTVVGGDEPDEFLQIKSLVLDGPAALDGKMETGDVIVSVNDTCVLGHTHAQVVKIFQSIPIGASVDLELCRGYPLPF DPDgihrd

3. TAX Interacting Protein 1 (TIP1):

Acc#:AF028823.2

GI#: 11908159

-   -   Construct: TIP1, PDZ domain 1 of 1-pGEX-3×        -   Primers: 399& 400        -   Vector Cloning Sites(5′/3′): Bam H1/EcoR1        -   Insert Cloning Sites(5′/3′): BamH1/EcoR1        -   aa 1-aa 107

(SEQ ID NO: 289) giQRVEIHKLRQGENLILGFSIGGGIDQDPSQNPFSEDKTDKGIYVTRVSEGGPAEIAGLQIGDKIMQVNGWDMTMVTHDQARKRLTKRSEEVVRLLVTR QSLQnss

-   -   Construct: TIP1-pGEX-3×        -   Primers: 1319& 1320        -   Vector Cloning Sites(5′/3′): Bam H1/EcoR1        -   Insert Cloning Sites(5′/3′): BamH1/EcoR1        -   aa 1-aa 128

(SEQ ID NO: 290) giqMSYIPGQPVTAVVQRVEIHKLRQGENLILGFSIGGGIDQDPSQNPFSEDKTDKGIYVTRVSEGGPAEIAGLQIGDKIMQVNGWDMTMVTHDQARKRLTKRSEEVVRLLVTRQSLQKAVQQSMnss

-   -   construct: TIP1-MIN        -   Primers: 2753& 2762        -   Vector Cloning Sites(5′/3′): EcoR1/EcoR1        -   Insert Cloning Sites(5′/3′): EcoR1/EcoR1        -   aa 1-aa 129

(SEQ ID NO: 291) agilEMSYIPGQPVTAVVQRVEIHKLRQGENLILGFSIGGGIDQDPSQNPFSEDKTDKGIYVTRVSEGGPAEIAGLQIGDKIMQVNGWDMTMVTHDQARKRLTKRSEEVVRLLVTRQSLQKAVQQSMLS

-   -   Construct: TIP1-CD5γ        -   Primers: 2584& 2585        -   Vector Cloning Sites(5′/3′): Bam H1/Bam H1        -   Insert Cloning Sites(5′/3′): BamH1/Bam H1        -   aa 1-aa 122

(SEQ ID NO: 292) adPGQPVTAVVQRVEIHKLRQGENLILGFSIGGGIDQDPSQNPFSEDKTDKGIYVTRVSEGGPAEIAGLQIGDKIMQVNGWDMTMVTHDQARKRLTKRSEEVVRLLVTRQSLQKAVQQSdpe

D. GST Fusion Protein Production and Purification

The constructs using pGEX-3× expression vector were used to make fusionproteins according to the protocol outlined in the GST Fusion System,Second Edition, Revision 2, Pharmacia Biotech. Method II and wasoptimized for a 1 L LgPP.

Purified DNA was transformed into E. coli and allowed to grow to anOD₆₀₀ of 0.4-0.8 (600λ). Protein expression was induced for 1-2 hours byaddition of IPTG to cell culture. Cells were harvested and lysed. Lysatewas collected and GS4B beads (Pharmacia Cat# 17-0756-01) were added tobind GST fusion proteins. Beads were isolated and GST fusion proteinswere eluted with GEB II. Purified proteins were stored in GEB II at −80°C.

Purified proteins were used for ELISA-based assays and antibodyproduction.

E. IgG Fusion Protein Production and Purification

The constructs using the CD5gamma or Peak10IgG expression vectors wereused to make fusion protein. Purified DNA vectors were transfected into293 EBNA T cells under standard growth conditions (DMEM+10% FCS) usingstandard calcium phosphate precipitation methods (Sambrook, Fritsch andManiatis, Cold Spring Harbor Press) at a ratio of ˜1 ug vector DNA for 1million cells. This vector results in a fusion protein that is secretedinto the growth medium. Transiently transfected cells are tested forpeak expression, and growth media containing fusion protein is collectedat that maxima (usually 1-2 days). Fusion proteins are either purifiedusing Protein A chromatography or frozen directly in the growth mediawithout addition.

EXAMPLE 5 TIP-1 Specifically Binds to Oncogenic E6 Proteins A. Abstract

An experiment was conducted to demonstrate and confirm that PDZ domainswould only recognize the C-termini of full-length oncogenic HPV E6proteins and not non-oncogenic E6 variants. This validates the method ofusing peptides representing the PL sequences of E6 proteins by asking ifthe PDZ binding can be reproduced using full length E6 fusion proteins.

Briefly, GST-E6 fusion proteins were constructed as described in Example3 corresponding to the full length protein sequence of E6 from HPV18(oncogeneic) and HPV11 (non-oncogenic). Using a modified ELISA assay,binding of a TIP-TIP-IgG fusion protein (two copies of the TIP-1 PDZdomain fused to the hIgG constant region, purification of fusion proteinpartially described in Example 4) to these two E6 variants was assessed.

A subsequent experiment is also shown to demonstrate that the assay forbinding to E6 using GST-Tip or GST-Magi fusion proteins is notsignificantly affected by incubation at 4° C. or room temperature (RT).

B. Modified ELISA Method

Reagents and Materials

Nunc Polysorp 96 well Immuno-plate (Nunc cat#62409-005)

-   -   (Maxisorp plates have been shown to have higher background        signal)

PBS pH 7.4 (Gibco BRL cat#16777-148) or

-   -   AVC phosphate buffered saline, 8 gm NaCl, 0.29 gm KCl, 1.44 gm        Na₂₁HPO4, 0.24 gm KH₂PO4, add H2O to 1 L and pH 7.4; 0.2 micron        filter

2% BSA/PBS (10 g of bovine serum albumin, fraction V (ICN Biomedicalscat#IC15142983) into 500 ml PBS

Goat anti-GST mAb stock @ 5 mg/ml, store at 4° C., (Amersham Pharmacia

-   -   cat#27-4577-01), dilute 1:1000 in PBS, final concentration 5        ug/ml

Wash Buffer, 0.2% Tween 20 in 50 mM Tris pH 8.0

TMB ready to use (Dako cat#S1600)

1M H₂SO₄

12w multichannel pipettor,

50 ml reagent reservoirs,

15 ml polypropylene conical tubes

anti E6HPV18 antibody(OEM Sciences)

Anti-hIgG-HRP (Biomeda)

Protocol

-   1) Coat plate with 5 ug/ml GST-E6 fusion protein, O/N @ 4° C.-   2) Dump proteins out and tap dry-   3) Blocking—Add 200 ul per well 2% BSA/PBS, 2 hrs at 4° C.-   4) Prepare PDZ proteins (50:50 mixture of supernatant from    TIP-TIP-IgG transfection and 2% BSA/PBS)-   5) 3× wash with cold PBS-   6) Add PDZ protein prepared in step 7 or anti-E6 Ab at 1 ug/ml in 2%    BSA/PBS (or anti-GST Ab as control).-   7) 3× wash with cold PBS-   8) Add appropriate concentration of enzyme-conjugated detection Ab    (anti-hIgG-HRP, anti-goat-HRP, or anti-mouse-HRP) 100 ul per well on    ice, 20 minutes at 4° C.-   9) Turn on plate reader and prepare files-   10) 5× wash with Tween wash buffer, avoiding bubbles-   11) Using gloves, add TMB substrate at 100 ul per well    -   incubate in dark at room temp    -   check plate periodically (5, 10, & 20 minutes)    -   take early readings, if necessary, at 650 nm (blue)    -   at 30 minutes, stop reaction with 100 ul of 1M H₂SO₄    -   take final reading at 450 nm (yellow)

C. Results of Binding Experiments

TIP-1, a representative PDZ domain that binds most oncogenic E6 PLs(EXAMPLE 2), is able to specifically recognize PLs from full lengthoncogenic E6 variants (HPV1 8-E6) without binding to non-oncogenicvariants (HPV11-E6; FIG. 1). Furthermore, even unpurified TIP-TIP-IgGfusion protein is able to recognize GST-HPV18E6 fusion protein at levelscomparable to an antibody generated against HPV18-E6. Antibodies againstGST were used to confirm that the GST-HPV18E6 and GST-HPV11E6 wereuniformly plated (data not shown).

Furthermore, this assay is robust and the off rates are stable enoughthat the incubation steps of this assay can be performed at 4° C. or RT.Little difference in signal is seen between the two temperatures foreither GST-Magi1 of GST-TIP1 binding to E6 (FIG. 2).

E6 activity may be further determined by its ability to bind DNA, or toallow degradation of p53 in the presence of a lysate, Zn2+ binding, etc.

EXAMPLE 6 EC50 Determinations for PDZ Domain Interactions with HPV16 E6

Using the G-assay described above, several GST-PDZ domain fusionproteins were tested to determine their relative binding strength to thePL of the HPV16 E6 protein. Peptide corresponding to the PL of HPV16 E6was titrated against a constant amount of GST-PDZ domain fusion and theresults are shown below. These results demonstrate that although anumber of PDZ domains can bind the E6 protein from HPV16, the firstfunctional domain of MAGI1 (domain 2 in this specification) binds themost tightly, making it the most suitable for diagnostic purposes. Thisis unexpected, especially in conjunction with MAGI1 being the only PDZdomain containing protein demonstrated to bind to all classes ofoncogenic E6 proteins identified. Together, these suggest that MAGI1 isa useful capture/detection agent for oncogenic HPV infections.

TABLE 7 EC50 values for HPV16 E6 protein with various PDZ domains RNAexpression (Cervical PDZ gene EC50^(a) [uM] cell lines) Magi1C (PDZ2)0.056 ++ Magi3 (PDZ1) 0.31 neg. SAST1 KIAA 0.58 neg. TIP1 0.75 +++VARTUL 0.94 + DLG1 (PDZ2) ND ++++ PSD95 (PDZ1-3) 1.0 ND SAST2 1.2 NDDLG2 (PDZ3) 1.6 ND DLG3 (PDZ1-2) 3.8 ND PSD95 (PDZ2) 6.8 ND SIP1 (PDZ1)7.5 ND Table 7 legend: ND = not done.

EXAMPLE 7 Production of Antibodies Against Purified E6 Fusion Proteinsfrom HPV18

In order to achieve the added benefits of a sandwich ELISA-baseddiagnostic for oncogenic HPV infection, high-affinity antibodiesspecific to E6 proteins should be generated. Ideally, monoclonalantibodies could be generated from these animals to have a continuallyrenewable resource for the diagnostic.

Balb/c mice were injected with 25 ug of bacterially purified GST-HPV18E6protein at 5 day intervals (Josman Labs). Sera from these mice werecollected 3 days after each injection of antigen and tested forreactivity with GST-HPV18E6 (the immunogen) or GST alone following antiGST-depletion (Pharmacia protocol). The results using sera collected atday 28 are shown in FIG. 3. The sera from this mouse reacts withbacterially purified GST-HPV18-E6 protein but do not react with GSTalone. This animal is a good candidate from which to generate amonoclonal antibody by standard methods.

EXAMPLE 8 Pathogen PL Proteins

Many other proteins from pathogens can be detected using proteins orcompounds directed at detection of a PDZ:PL interaction. Table 8contains some exemplary proteins that could be detected using technologydisclosed herein, but is not meant to be limiting in any manner.

TABLE 8 Example Pathogens amenable to PDZ:PL diagnostics Gi or ACC PL/Pathogen Protein number PDZ Adenovirus E4 19263371 PL Hepatitis BProtein X 1175046 PL virus Human T TAX 6983836 PL Cell Leukemia VirusHerpesvirus DNA polymerase 18307584 PL Herpesvirus US2 9629443 PL

EXAMPLE 9 Quantification of Endogenous E6 Protein in Cells Infected withHPV16 A) Abstract:

Experiments were designed and performed to determine quantities ofendogenous E6 protein in HPV16 infected cervical cancer cell lines.Results demonstrate that HPV16 infected cervical cancer cell linescontain in the order of 10,000 to 100,000 molecules E6. From thisfinding is concluded, that E6 protein can be used as a diagnostic orprognostic marker for cellular HPV infection. Use of protein degradationpathway inhibitors may facilitate such an assay.

B) Methods: Immunoprecipitation of E6 Protein:

HPV16-infected cervical cancer cell lines SiHa and CasKi are washed withcold PBS and resuspended in HEPES lysis buffer (50 mM HEPES pH 7.4, 150mM NaCl, 10% glycerol, 0.5% triton X-100, 1 mg/ml BSA, one pelletprotease inhibitor cocktail (Roche), and 1 mM PMSF) at 2×10⁷ cells/ml.Lysis proceeds on ice for 30 min. and lysates are cleared bycentrifugation at 14,000×g for 5 minutes at 4° C. E6 proteins areimmunoprecipitated with a mouse anti-E6 antibody (clone 6F4) and proteinG beads (Pharmacia, Piscataway, N.J.). After 2 hours incubation at 4° C.with rotation, beads are washed 3 times with washing Buffer [50 mM HEPESpH 7.4, 150 mM NaCl, 10% glycerol, 0.1% Triton X-100, protease inhibitorcocktail (CALBIOCHEM), 1 mM PMSF]. Pellets are resuspended in SDS-PAGEsample buffer and analyzed by immuno blotting using 6F4 anti-E6 antibodyand anti-mouse-IgG-HRP conjugated (Jackson Immuno Research).

Detection of E6 Protein from Cervical Cancer Cell Lysates by WesternTechnology:

SiHa and CasKi cervical cancer cell lines were lysed at 2×10⁷ cells/mlin lysis buffer 30 min. on ice. Lysates corresponding to approx. 10⁶cells are immediately resolved on a 12% SDS-PAGE gel followed bytransfer to a PVDF membrane. E6 proteins were detected with 6F4 anti-E6HPV16 antibody and anti-mouse-IgG-HRP conjugated (Jackson ImmunoResearch).

C) Results:

To determine the apparent molecular weight of endogenous E6 protein aspresent in cervical cancer cells upon infection with HPV16 and to ensurethat a anti E6 monoclonal antibody-specific band seen in PAGE representsviral E6 protein, 293 EBNA-T cells were transfected with a constructexpressing untagged E6 protein of HPV type 16. Cell lysates wereprepared of those cells, and HPV infected SiHa cervical cancer cells. E6protein from both lysates (transfected and HPV infected) wasimmunoprecipitated by use of an anti E6-specific monoclonal antibody.Both lysates were analyzed side by side using PAGE technology (FIG. 6).The E6-specific band obtained for transfected E6 migrates in PAGE at thesame level as the anti E6 antibody specific band from SiHa cervicalcancer cell lines, thus most strongly suggesting that the productimmunoprecipitated with anti E6-specfic monoclonal antibody representviral E6 protein. Using the specific E6 monoclonal antibody, a band ofthe same size was detected in HPV16 infected cervical cancer cell typeCasKi (FIG. 7).

In a different experimental procedure, endogenous viral E6 protein ofHPV16 infected cervical cancer cell line SiHa and CasKi was directlydetected from their cell lysates (FIG. 7). Bands that were dependent onE6-specific monoclonal antibody ran in the same way as the band forcells transfected with E6 encoding vector.

To test, whether E6 in vivo stability can be enhanced by selectivelyblocking proteasome involved in protein degradation, cell lysates ofsome samples were treated with proteasome inhibitor MG132. In thosesamples, the E6 specific band is about 2-3 times more intense. Thisdemonstrates, that addition of an appropriate mixture of proteindegradation pathway inhibiting agents can be used to increase the signalspecific to E6 protein by augmenting its accumulation temporarily incells.

Quantities of E6 protein in lysates were measured by comparingE6-Specific signal in PAGE with signals obtained by MBP-E6 (HPV16)fusion protein loaded onto the same gel. In some cases, MBP-E6 fusionprotein was digested with factor X to release the E6 portion only.Signal intensity comparison studies demonstrated, that cervical cancerderived cell lines injected with HPV16 (SiHa, CasKi) contain E6 at aconcentration of 0.3 to 3 ng per 1×10⁶ cells. It is concluded, thatquantities and stability of E6 are such that detection by an E6-specific(ELISA-) assay will be feasible.

EXAMPLE 10 Oncogenic E6-PL-Detector Molecules Bind SelectivelyEndogenous HPV6-E6 Proteins Present in Cell Lysates and can be used toSeparate Endogenous E6 Protein from Other Components Present in a CellLysate A) Abstract

Experiments were undertaken to test, whether oncogenic E6-PL-detectorwill selectively bind endogenous E6 of cells transfected with E6encoding vector. Moreover, it was tested whether the oncogenicE6-PL-detector can be used to separate E6 from other molecules in thecell lysate subsequent to binding. Findings demonstrate that oncogenicE6-PL-detector, is selective and can be applied to separate E6 proteinfrom the complex mixture of cell lysate molecules.

B) Methods

Pull Down of E6 Protein with Recombinant PDZ Proteins:

GST-PDZ fusion proteins (i.e. Magi1 PDZ domain #1, Syn2 bp, Magi3 PDZdomain # 1, Tip1, PSD-95 PDZ domain # 2, and SAST1 were tested in pulldown experiments. Briefly, 10 ug recombinant GST-PDZ proteins wereincubated with 30 ul of glutathione-sepharose beads in 1 ml of buffer[50 mM HEPES pH 7.4, 150mM NaCl, 10% glycerol, 0.1% Triton X-100,protease inhibitor cocktail, 1 mM PMSF] for 1 h at 4° C. with rotation.Subsequently, cell lysates of 10⁷ 293 cells transiently transfected witheither pMKit-HA-HPV16-E6 or pMKit-HA vector alone were incubated withthe beads bound to PDZ proteins for 3 h at 4° C. with rotation. Beadswere washed and analyzed in 12% SDS-PAGE gel electrophoresis followed byWestern blotting. Membranes were probed with biotin conjugated anti-HAantibodies (clones 3F10, or 12CA5, Boehringer Mannheim) andHRP-Streptavidin (Zymed).

Alternatively, cell lysates from 293 cells transiently transfected withpmKit-HA, pmkit-HPV16-HA-E6 or pmKit-HA-HPV16 E6-□PL, were incubatedwith recombinant GST-Magi1-PDZ domain1 protein and immobilized onglutathione-sepharose beads and bound fractions were immunoblotted withanti HA antibodies. In parallel, lysates were immunoprecipitated anddetected with anti-HA antibodies.

C) Results

G-assay PDZ-E6-PL binding studies and the determination of experimentalbinding affinities of the E6-PDZ interactions suggested candidate PDZdomains to be tested for the engineering of an oncogenic E6-PL-detector.In a “pull down” experiment, five different PDZ domains (Tip1; Magi1domain 1; Sast2; Psd95 domain 2; Synaptojanin-2 binding protein) weretested for pull down of endogenous over expressed E6 from cell lysate.Lysates of cells transfected with HA-tagged E6 HPV-16 were incubatedwith GST-PDZ fusion protein representing the above PDZ domains bound toSepharose beads (FIG. 5). Control cell samples were transfected with HAexpressing constructs. Detection with anti HA monoclonal antibodydemonstrates, that E6 is selectively pulled out of cell lysates via thePDZ domain represented by the oncogenic E6-PL-detector of all fiveGST-PDZ proteins tested (Tip1; Magi1 domain 1; Sast2; Psd95 domain 2;Synaptojanin-2 binding protein). Results shown in FIG. 5B demonstratethat Magi 1-PDZ domain 1 associates with HA-E6 but not with HA-E6ΔPL(lacking the 3 C-terminal amino acids). This method can be used todetermine, whether a particular PDZ domain has the capacity of specificE6 binding. The conclusion is made, that competition by potentially PDZbinding proteins represented by the complex mixture of cell lysates andE6 for binding to PDZs can be shifted towards selective binding of E6 byappropriate choice of the specific PDZ domain that constitutes theoncogenic E6-PL detector.

EXAMPLE 11 Endogenous E6 Protein of HPV Infected Cervical Cancer CellLines can be Detected in a Sandwich ELISA Via the Oncogenic E6-PLDetector Molecule A) Abstract:

Experiments are described, in which the oncogenic E6-PL detector is usedto selectively detect presence of E6 protein in HPV infected cells via asandwich ELISA. The specific capturing of oncogenic E6 but notnon-oncogenic E6 demonstrates that the PDZ based oncogenic E6-PLdetector can be applied for a E6 detection based diagnostic test for HPVinfection and/or cervical cancer test.

B) Methods:

Sandwich type 1 ELISA: Anti-E6 antibody is coated onto a 96-wellPolysorp or Maxysorp ELISA plate at 5 ug/ml in PBS (100 ul/well)overnight at 4° C. Plates were washed with PBS and blocked with 200 ulPBS/2% BSA for 2 hours at 4° C. Cell lysates diluted in PBS/2% BSA areadded and incubated at room temperature for 1 hour. After 3 washes withPBS, 100 ul of oncogenic E6 detector (for example MAGI1-MAGI1-IgG orGST-MAGI1-PDZ1) at 5 ug/ml was added in PBS/2% BSA, and plates areincubated at room temperature for 45 min. Plates are then washed 3 timeswith PBS and incubated with anti-hIgG-HRP (Jackson Immuno Research) oranti-GST-HRP (Pharmacia) at the appropriate concentration in PBS/2% BSAat room temperature for 45 minutes. After 5 washes with 50 mM Tris/0.2%Tween-20, plates were incubated with 100 ul/well TMB substrate (DakoIndustries). The colorimetric reaction is stopped at appropriate times(usually after 20 minutes) by addition of 100 ul of 0.1 M H₂SO₄ andplates read at A₄₅₀ nm in an ELISA plate reader.

In a variant of sandwich 1 ELISA, cell lysates were preincubated withoncogenic E6 detector at 2.5-5 ug/ml final concentration, for 1-2 hoursat 4° C., prior to adding to the anti-E6 antibody coated plate.

Sandwich type 2 ELISA: In sandwich 2, reagents and procedures mostlycorrespond to those used in sandwich 1. In contrast to sandwich 1, 100ul of oncogenic E6 detector is coated onto the ELISA plate and theanti-E6 antibody is used for detection of oncogenic detector-bound E6,followed by anti-mouse IgG-HRP (Jackson Immuno Research). In a modifiedversion of sandwich 2, biotinylated reagents (anti-E6 antibody oroncogenic detector) will be used followed by streptavidin-HRP to furtherdiminish background and to increase sensitivity.

C) Results:

A sandwich ELISA was conceived in two different variations. In Type 1sandwich ELISA, E6 protein present in cell lysates in captured byE6-specific monoclonal antibody, and detection of specifically oncogenicvariants occurs via the oncogenic E6-PL detector. In the type 2 ELISAset up, oncogenic E6 protein is captured via the oncogenic E6-PLdetector to the solid phase and E6 detection occurs via a specific E6antibody or another E6 binding specific agent like nucleic acid basedbinding compounds, chemicals binding E6, E6 binding proteins or acombination of those compounds. Cells were lysed directly on a tissueculture plate and lysates were precleared by centrifugation frominsoluble components. Lysates were preincubated at 4° C. with oncogenicE6-PL detector, a fusion protein of GST and Magi1 PDZ domain #1.Subsequently, lysates were loaded onto E6-specific antibody coated ELISAplates. Detection occurred via addition of HRP conjugated GST-specificantibody and addition of the HRP substrate TMB after appropriate washesbetween different incubation steps. Detection signal is constituted by acolorimetric change that is quantified using absorbance measurements at450 nm.

Results obtained from a type 1 ELISA assay are shown. HPV16-E6 of overexpressing E6 transfected 293 EBNA-T cells and of HPV16 infectedcervical cancer derived cell lines was detected. For HPV infected cells,the detection limit is at approximately 250,000 cells (FIG. 8). It ispredicted, that background reduction, detection signal enhancement andE6:PDZ binding enhancement will increase sensitivity to 25,000 cells orless. Background reduction can be achieved by optimizing choice andconcentrations of all components in the system, as well as by additionalcomponent purification or addition of size exclusion or filteringprocedures. Detection signal can be enhanced by use of more sensitivedetection systems, for example luminescence based technologies. E6:PDZbinding can be enhanced by modifying the PDZ base of the oncogenic E6-PLdetector, and by treating the E6 containing lysates with phosphatases,thus freeing all E6-PL sites from any phosphate that might interferewith, diminish or abrogate E6-P1-specific binding to the oncogenic E6-PLdetector.

EXAMPLE 12 Endogenous E6 Protein of HPV Infected Cervical Cancer CellLines Can be Detected Via a Membrane Bound Oncogenic E6-PL Detector.Membrane Based Detection can be Used to Enhance Sensitivity of OncogenicE6-PL Detector Based Assay. A) Abstract:

Experiments were conducted to demonstrate that the cervical cancer ELISAtest types 1 and 2 can be performed using a membrane based format. Inthe membrane-based form of the cervical cancer diagnostic kit, theprinciples of the traditional ELISA based sandwich 1 and 2 aremaintained, especially with regard to the capturing or detection ofexclusively the oncogenic forms of E6. Sensitivity is found to belargely increased in the membrane based assay versus the traditionalELISA.

B) Methods:

Preblock 12 well corning plates (tissue culture treated with lid,polystyrene, 22 mm well diameter) with 2 ml PBS/2% BSA and then rinse 3×with 2 ml PBS Spot nitrocellulose membrane with 2 ul GST-Magi1 d 1solution(88.6, 0.17 mg/ml) using 2 ul pipetman (duplicate spots in 1×1.5cm membrane, transblot, transfer medium, supported nitrocellulosemembrane, catalog no. 162-0097 (0.2 uM), Lot No. 8934). Allow to air dryfor 5-10 minutes.

Hydrate membrane with 1 ml PBS for a couple of minutes in plate.

Block membrane in each well with 1 ml PBS/2% BSA for 30 minutes at roomtemperature while rocking

Wash 3× with PBS ˜5-10 minutes/wash, 1 ml/wash, aspirate directly firstwash. OK to wash at room temperature.

Incubate membrane with cell lysate, ˜300 ul, 3 million cells total, for30 minutes at room temperature (rock solutions). Also perform 1:10dilutions (3 million, 300K, 30K, 3K) in PBS/2% BSA (33.33 ul sample, 300ul PBS/2% BSA)

Wash 3× with PBS, 3-5′/wash, all at 4 C, 1 ml/wash.

Incubate membrane with anti-E6 (6F4) for 30 minutes at 4° C. (1:5000dilution, or 1:50 of 1:100 6F4 in PBS/2% BSA). (Need 0.4 ml/well, andfor 36 wells need 16 ml a) 1) 320 ul of 1:100 6F4, 15.68 ml PBS/2% BSA.

Wash 3× with PBS, 4° C., ˜5-10 minutes/wash.

Incubate with HRP-anti-mouse (1:1000) for 30′ at 4° C. whilerocking(HRP-anti-Mouse Ig Horseradish peroxidase linked whole antibodyfrom Sheep, Amersham, NA93 IV, lot 213295. Use 400 ul per well. For 36wells would need a) 16 ul HRP-anti-mouse, 16 ml PBS/2% BSA

Wash 5× with PBS at 4° C., ˜5-10 minutes rocking/wash, last wash 10minutes. Then aspirate last wash, and add 1 ml fresh PBS to each well.

Develop with ECL+ system in Petri dish and expose in Kodak film.

C) Results:

In a sandwich type 2 setup, GST-MAGI1 oncogenic E6-PL detector wasspotted on a membrane and decreasing quantities of HPV11 and HPV16MBP-E6 fusion proteins were added for binding. Detection with E6specific antibodies clearly demonstrated specificity of signal foroncogenic (HPV16), but not non-oncogenic E6 (HPV11). Upon longerexposure (5 minutes), HPV16 MBP-E6 quantities of 0.1 nanogram total werereadily detectable (FIG. 9, top).

In the same experiment, lysates of HPV16-E6 transfected cells and mocktransfected cells were applied to a membrane based S2 test. E6-specificsignal was obtained only for the E6 expressing cells, not for mocktransfected cells (FIG. 9, bottom). These results clearly demonstratethat the membrane based cervical cancer test can be executed in amembrane-based format.

In a subsequent experiment, lysates of HPV infected cells were tested(FIG. 10). Clearly, only the HPV16-E6 expressing cells are yieldingsignal (SiHa and CasKi), but not the HPV negative but cervical cancerpositive cell line C33. E6-specific signal is obtained at 300.000 cells,indicating that an optimized form of this test may detect HPV-E6proteins of substantially lower cell numbers.

The present invention is not to be limited in scope by the exemplifiedembodiments which are intended as illustrations of single aspects of theinvention and any sequences which are functionally equivalent are withinthe scope of the invention. Indeed, various modifications of theinvention in addition to those shown and described herein will becomeapparent to those skilled in the art from the foregoing description andaccompanying drawings. Such modifications are intended to fall withinthe scope of the appended claims.

All publications cited herein and priority documents cited in theApplicant Data Sheet are incorporated by reference in their entirety andfor all purposes.

TABLE 9 Seq Id name gi/acc domain 1 26s subunit p27 9184389 1 2 AF6 430993 1 3 AIPC 12751451  1 4 AIPC 12751451  2 5 AIPC 12751451  3 6AIPC 12751451  4 7 alpha actinin-2 2773059 1 associated LIM protein 8APXL-1 13651263  1 9 Atrophin-1 2947231 1 Interacting Protein 10Atrophin-1 2947231 2 Interacting Protein 11 Atrophin-1 2947231 3Interacting Protein 12 Atrophin-1 2947231 4 Interacting Protein 13Atrophin-1 2947231 5 Interacting Protein 14 Atrophin-1 2947231 6Interacting Protein 15 CARD11 12382772  1 16 CARD14 13129123  1 17 CASK3087815 1 18 Connector Enhancer 3930780 1 19 Cytohesin Binding Protein3192908 1 20 Densin 180 16755892  1 21 DLG1  475816 1 22 DLG1  475816 223 DLG1  475816 3 24 DLG2 12736552  1 25 DLG2 12736552  2 26 DLG212736552  3 27 DLG5 3650451 1 28 DLG5 3650451 2 29 DLG6, splice variant1 14647140  1 30 DLG6, splice variant 2 AB053303 1 31 DVL1 2291005 1 32DVL2 2291007 1 33 DVL3 6806886 1 34 ELFIN 1 2957144 1 35 ENIGMA  5616361 36 ERBIN 8923908 1 37 EZRIN Binding Protein 50 3220018 1 38 EZRINBinding Protein 50 3220018 2 39 FLJ00011 10440352  1 40 FLJ1121511436365  1 41 FLJ12428 BC012040 1 42 FLJ12615 10434209  1 43 FLJ200757019938 1 44 FLJ21687 10437836  1 45 FLJ31349 AK055911 1 46 FLJ32798AK057360 1 47 GRIP 1 4539083 1 48 GRIP 1 4539083 2 49 GRIP 1 4539083 350 GRIP 1 4539083 4 51 GRIP 1 4539083 5 52 GRIP 1 4539083 6 53 GRIP 14539083 7 54 GTPase Activating Enzyme 2389008 1 55 Guanine ExchangeFactor 6650765 1 56 HEMBA 1000505 10436367  1 57 HEMBA 1000505 10436367 2 58 HEMBA 1003117 7022001 1 59 HTRA3 AY040094 1 60 HTRA4 AL576444 1 61INADL 2370148 1 62 INADL 2370148 2 63 INADL 2370148 3 64 INADL 2370148 465 INADL 2370148 5 66 INADL 2370148 6 67 INADL 2370148 7 68 INADL2370148 8 69 KIAA0147 1469875 1 70 KIAA0147 1469875 2 71 KIAA01471469875 3 72 K1AA0147 1469875 4 73 KIAA0303 2224546 1 74 KIAA03137657260 1 75 KIAA0316 6683123 1 76 KIAA0340 2224620 1 77 KIAA03802224700 1 78 KIAA0382 7662087 1 79 KIAA0440 2662160 1 80 KIAA054514762850  1 81 KIAA0559 3043641 1 82 KIAA0561 3043645 1 83 KIAA06133327039 1 84 KIAA0751 12734165  1 85 KIAA0807 3882334 1 86 KIAA08584240204 1 87 KIAA0902 4240292 1 88 KIAA0967 4589577 1 89 KIAA09734589589 1 90 KIAA1095 5889526 1 91 KIAA1095 5889526 2 92 KIAA12026330421 1 93 KIAA1222 6330610 1 94 KIAA1284 6331369 1 95 KIAA13897243158 1 96 KIAA1415 7243210 1 97 KIAA1526 5817166 1 98 KIAA15265817166 2 99 KIAA1526 5817166 3 100 KIAA1620 10047316  1 101 KIAA163410047344  1 102 KIAA1634 10047344  2 103 KIAA1634 10047344  3 104KIAA1634 10047344  4 105 KIAA1634 10047344  5 106 KIAA1719 1267982 0 107KIAA1719 1267982 1 108 KIAA1719 1267982 2 109 KIAA1719 1267982 3 110KIAA1719 1267982 4 111 KIAA1719 1267982 5 112 KIAA1719 1267982 6 113 LIMMystique 12734250  1 114 LIM Protein 3108092 1 115 LIMK1 4587498 1 116LIMK2 1805593 1 117 LIM-RIL 1085021 1 118 LU-1 U52111 1 119 MAGI13370997 1 120 MAGI1 3370997 2 121 MAGI1 3370997 3 122 MAGI1 3370997 4123 MAGI1 3370997 5 124 MAGI1 3370997 6 125 MGC5395 BC012477 1 126 MINT12625024 1 127 MINT1 2625024 2 128 MINT3 3169808 1 129 MINT3 3169808 2130 MPP1  189785 1 131 MPP2  939884 1 132 MUPP1 2104784 1 133 MUPP12104784 2 134 MUPP1 2104784 3 135 MUPP1 2104784 4 136 MUPP1 2104784 5137 MUPP1 2104784 6 138 MUPP1 2104784 7 139 MUPP1 2104784 8 140 MUPP12104784 9 141 MUPP1 2104784 10 142 MUPP1 2104784 11 143 MUPP1 2104784 12144 MUPP1 2104784 13 145 NeDLG 10863920  1 146 NeDLG 10863920  2 147NeDLG 10863920  3 148 Neurabin II AJ401189 1 149 NOS1  642525 1 150novel PDZ gene 7228177 1 151 novel PDZ gene 7228177 2 152 Novel SerineProtease 1621243 1 153 Numb Binding Protein AK056823 1 154 Numb BindingProtein AK056823 2 155 Numb Binding Protein AK056823 3 156 Numb BindingProtein AK056823 4 157 Outer Membrane 7023825 1 158 p55T 12733367  1 159PAR3 8037914 1 160 PAR3 8037914 2 161 PAR3 8037914 3 162 PAR6 2613011 1163 PAR6 GAMMA 13537118  1 164 PDZ-73 5031978 1 165 PDZ-73 5031978 2 166PDZ-73 5031978 3 167 PDZK1 2944188 1 168 PDZK1 2944188 2 169 PDZK12944188 3 170 PDZK1 2944188 4 171 PICK1 4678411 1 172 PIST 98374330  1173 prlL16 1478492 1 174 prlL16 1478492 2 175 PSD95 3318652 1 176 PSD953318652 2 177 PSD95 3318652 3 178 PTN-3  179912 1 179 PTN-4  190747 1180 PTPL1  515030 1 181 PTPL1  515030 2 182 PTPL1  515030 3 183 PTPL1 515030 4 184 PTPL1  515030 5 185 RGS12 3290015 1 186 RGS3 18644735  1187 Rhophilin-like 14279408  1 188 Serine Protease 2738914 1 189 Shank 16049185 1 190 Shank 3 * 1 191 Shroom 18652858  1 192 SIP1 2047327 1 193SIP1 2047327 2 194 SITAC-18 8886071 1 195 SITAC-18 8886071 2 196 SSTRIP7025450 1 197 SYNTENIN 2795862 1 198 SYNTENIN 2795862 2 199 Syntrophin 1alpha 1145727 1 200 Syntrophin beta 2  476700 1 201 Syntrophin gamma 19507162 1 202 Syntrophin gamma 2 9507164 1 203 TAX2-like protein 32531161 204 TIAM 1 4507500 1 205 TIAM 2 6912703 1 206 TIP1 2613001 1 207 TIP22613003 1 208 TIP33 2613007 1 209 TIP43 2613011 1 210 X-11 beta 30055591 211 X-11 beta 3005559 2 212 ZO-1  292937 1 213 ZO-1  292937 2 214 ZO-1 292937 3 215 ZO-2 12734763  1 216 ZO-2 12734763  2 217 ZO-2 12734763  3218 ZO-3 10092690  1 219 ZO-3 10092690  2 220 ZO-3 10092690  3 221 HPV4 - E6 222 HPV 11-E6 223 HPV 20-E6 224 HPV 24-E6 225 HPV 28-E6 226 HPV36-E6 227 HPV 48-E6 228 HPV 50-E6 229 HPV 16-E6 230 HPV 18-E6 231 HPV31-E6 232 HPV 35-E6 233 HPV 30-E6 234 HPV 39-E6 235 HPV 45-E6 236 HPV51-E6 237 HPV 52-E6 238 HPV 56-E6 239 HPV 59-E6 240 HPV 58-E6 241 HPV33-E6 242 HPV 66-E6 243 HPV 68-E6 244 HPV 69-E6 245 HPV 26 246 HPV 53247 HPV 66 248 HPV 73 249 HPV 82 250 2548 (1054EF) 251 2549 (1058ER) 2522542 (1050EF) 253 2543 (1051ER) 254 2563 (1071EF) 255 2564 (1072ER) 2562565 (1073ERPL) 257 2560 (1074EF) 258 2561 (1075ER) 259 2562 (1076ERPL)260 2603 (1080EF) 261 2604 (1081ER) 262 2605 (1082ERPL) 263 2606(1083EF) 264 2607 (1084ER) 265 2608 (1085ERPL) 266 2612 (1086EF) 2672613 (1087ER) 268 2614 (1088ERPL) 269 2615 (1089EF) 270 2616 (1090ER)271 2617 (1091ERPL) 272 GST 273 1928 (654DL1 2F) 274 1929 (655DL1 2R)275 1453 (435BAF) 276 1454 (436BAR) 277 399 (86TAF) 278 400 (87TAR) 2791319 (TIP G5-1) 280 1320 (TIP G3-1) 281 2753 (1109TIF) 282 2762(1117TIR) 283 2584 (1080TIF) 284 2585 (1081TIR) 285 2586 (1082TIR) 2862587 (1083TIF) 287 DLG 1, PDZ d 2 of 3 288 MAGI 1, PDZ d2 of 6 289 TIP1290 TIP1-FL-pGEX 291 TIP1-Min 292 TIP1-CD5g 293 Magi1D2v2 294 Magi1D2v3295 Magi1D2v4 296 Magi1D2v5 297 Magi1D2v6 298 Magi1D2v7 299 Magi1D2v8300 Magi1D2v9 301 Magi1D2v10 302 Magi1D2v11 303 Magi1D2v12 304Magi1D2v13 305 Magi1D2v14 306 Magi1D2v15 307 Magi1D2v16 308 Magi1D2v17309 Magi1D2v18 310 Magi1D2v19 311 Magi1D2v20 312 Magi1D2v21 313Magi1D2v22 314 Magi1D2v23 315 Magi1D2v24 316 Magi1D2v25 317 Magi1D2v26318 Magi1D2v27 319 Magi1D2v28 320 Magi1D2v29 321 Magi1D2v30 322Magi1D2v31 323 Magi1D2v32 324 Magi1D2v33 325 Magi1D2v34 326 Magi1D2v35327 Magi1D2v36 328 Magi1D2v37 329 Magi1D2v38 330 Magi1D2v39

1. A method of detecting the presence of an oncogenic HPV E6 protein asample, said method comprising: contacting a sample suspected ofcontaining an oncogenic HPV E6 protein with a PDZ domain polypeptide;and detecting any binding of said oncogenic HPV E6 protein in saidsample to said PDZ domain polypeptide; wherein binding of said oncogenicHPV E6 protein to said PDZ domain polypeptide indicates the presence ofan oncogenic HPV E6 protein in said sample.
 2. The method of claim 1,wherein said PDZ domain polypeptide comprises the amino acids sequenceof Magi-1 PDZ domain
 2. 3. The method of claim 1, wherein said PDZdomain peptide binds to HPV E6 protein encoded by HPV strains 16, 18 and45.
 4. The method of claim 1, wherein sample is contacted with multiplePDZ domain polypeptides.
 5. The method of claim 1, wherein said PDZprotein is a fusion protein.
 6. A system for detecting the presence ofan oncogenic HPV E6 polypeptide in a sample, said method comprising: afirst and a second binding partner for an oncogenic HPV E6 polypeptide,wherein said first binding partner is a PDZ domain protein and at leastone of said binding partners is attached to a solid support.
 7. Thesystem of claim 6, wherein said second binding partner is an antibodyagainst said oncogenic HPV E6 polypeptide.
 8. The system of claim 7,wherein at least one of said binding partners is labeled.
 9. The methodof claim 6, wherein said PDZ domain protein comprises the amino acidsequence of Magi-1 PDZ domain
 2. 10. A method for determining if asubject is infected with an oncogenic strain of HPV, said methodcomprising: detecting the presence of oncogenic HPV E6 protein in asample from said subject using an oncogenic HPV E6 protein-binding PDZprotein, wherein the presence of oncogenic HPV E6 protein indicates thatthe subject is infected with an oncogenic strain of HPV.
 11. The methodof claim 10, wherein said detecting step further comprises detecting thepresence of said oncogenic HPV E6 protein using an antibody thatspecifically binds to said oncogenic HPV E6 protein.
 12. The method ofclaim 10, wherein said sample is a cervical scrape, biopsy, or lavage.13. The method of claim 12, wherein said method is an ELISA or asandwich assay.
 14. The method of claim 10, wherein said sample isprepared in the presence of a proteasome inhibitor.
 15. The method ofclaim 10, wherein said method is a performed as part of a test forcervical cancer.
 16. A kit for testing for the presence of oncogenic HPVE6 protein, the kit comprising first and second binding partners forsaid oncogenic HPV E6 protein, wherein said first binding partner is aPDZ domain protein.
 17. The kit of claim 14, wherein at least one of thebinding partners is attached to a solid support.
 18. The kit of claim16, wherein said solid support is a test strip.
 19. The kit of claim 16,wherein said second binding partner is an antibody.
 20. The kit of claim16, further comprising instructions for detecting the presence of anoncogenic HPV E6 protein in a sample.